Biomarker for her2-positive cancer and anti-her2 therapy and applications thereof

ABSTRACT

The present invention provides MEL-18, which is a biomarker for human epidermal growth factor receptor2 (HER2)-positive cancer and anti-HER2 therapy, and a use thereof. According to the present invention, MEL-18 is a prognostic factor or predictor for a response of subjects to an anti-HER2-targeted drug in HER2-POSITIVE cancer and may be used in companion diagnostics for HER2-targeted drugs in subjects with HER2-positive cancer. Therefore, HER2-positive cancer may be more effectively treated by overcoming resistance to HER2 therapeutic agents and enhancing therapeutic efficacy by determining whether ADAM10/17 inhibitors are administered or co-administered with HER2-targeted drugs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Rule 53(b) Divisional of U.S. application Ser. No.16/491,993 filed Sep. 6, 2019 which is a National Stage of InternationalApplication No. PCT/KR2018/002791 filed Mar. 8, 2018, claiming prioritybased on Korean Patent Applications No. 10-2017-0029627, filed Mar. 8,2017 and 10-2017-0042224 filed Mar. 31, 2017. The contents of all of theabove-identified applications are incorporated herein by reference intheir entireties.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The content of the electronically submitted sequence listing, file name:Q287180_Sequence_Listing_As_Filed.XML; size 36.8 kilobytes; and date ofcreation: Apr. 27, 2023, is incorporated herein by reference in itsentirety.

TECHNICAL FIELD

The present invention relates to a biomarker for human epidermal growthfactor receptor2 (HER2)-positive cancer and anti-HER2 therapy, and a usethereof.

BACKGROUND ART

Although a drug is taken properly according to the doctor'sprescription, it may have different actions depending on a person, andit has been reported that about 2 million patients have experienced drugside effects, and 0.5% of these patients died from the drug sideeffects. In addition, only one third of patients prescribed drugsexhibit a desired therapeutic effect, and the report that the leadingcause of death in the United States results from adverse drug reactionsis an example clearly showing the blind spot of “one size fits all”medication. The biggest reason for differences in drug response amongindividuals is a “genetic factor.” Since different persons havedifferent genes, they have a difference in the amounts of enzymes andproteins in the body, and thus also have a difference in drugmetabolism. Such interpersonal diversity makes it difficult to estimatea drug effect and stability in a specific patient, and therefore, toovercome this problem, a personalized medical approach based on thegenetic information of each patient is required.

MEL-18 is a protein of the polycomb group (PcG) belonging topolycomb-repressive complex 1 (PRC1). PcG proteins are importantepigenetic regulators which consist of two multicomplexes: one ispolycomb-repressive complex 2 (PRC2), which is an initiation complex ofa silent gene, catalyzing histone 3 tri-methylation in lysine-27(H3K27me3); and the other is PRC1, which recognizes H3K27me3 andmaintains gene silencing via various mechanisms including histone H2Amono-ubiquitination on lysine-119 (H2AK119ub) and blockade oftranscriptional initiation machinery (1-3). While MEL-18 homolog BMI-1and the core components of PRC1 have been well established, the role ofMEL-18 in PRC1/2-mediated epigenetic gene silencing is poorlyunderstood. Previous studies showed that MEL-18 is associated with cellproliferation, angiogenesis, and inhibition of cancer stem cells (CSCs)and epithelial mesenchymal transition (EMT) in breast cancer (4, 11-14).In addition, it has been reported that MEL-18 loss mediates the decreasein estrogen receptors (ERs), hormone independence and tamoxifenresistance (15), leading to association with triple-negative breastcancer (TNBC), and also revealed that MEL-18 loss plays a decisive rolein the more aggressive progression of breast cancer.

Breast cancer having HER2 amplification or overexpression, accountingfor 15 to 25% of all types of breast cancer, is considered an aggressivephenotype (16, 17). HER2 (called ErbB/HER) is a member of the epidermalgrowth factor receptor family of receptor tyrosine kinases (RTKs). SinceHER2 has no identified ligand but is present in a suitable structure forhomo- or hetero dimerization with other ErbB family members, it inducesvarious types of oncogenic signaling associated with cell proliferation,survival, CSC and metastasis (18-21). Over the past several decades,HER2 has been considered a major therapeutic target for HER2-positive(HER2+) breast cancer (16, 18). Trastuzumab, which is a humanizedmonoclonal antibody against the HER2 extracellular domain, is the mostcommon drug for HER2+ breast cancer patients and has received FDAapproval (22), but only 30% of metastatic breast cancer patients respondto treatment with trastuzumab alone, and many patients acquireresistance to continuous treatment (23-26). There are several mechanismsof trastuzumab resistance, including target alteration (27, 28), ligandproduction, dimerization between ErbB family members (29-31), downstreammutation (32), and bypass signaling (33). Particularly,heterodimerization between ErbB family receptors is the main cause ofbypassing the trastuzumab inhibitory effect against HER2 (34).Therefore, various strategies for overcoming trastuzumab resistanceusing target drugs for ErbB signaling such as lapatinib (35) andpertuzumab (36) have been attempted. However, there is still a demandfor complete understanding of trastuzumab resistance and new therapeuticstrategies for HER2-positive cancers.

DISCLOSURE Technical Problem

To solve the conventional problems, the inventors had continuouslystudied to identify that a tumor suppressor MEL-18 plays a pivotal rolefor mediating responses to HER2-targeted drugs against HER2-positivecancers and to suggest a new therapeutic strategy for HER2-positivecancers.

Specifically, it was confirmed that MEL-18 is amplified only in almosthalf of HER2+ cancer patients, and it was newly found that MEL-18inhibits resistance to HER2-targeted drugs, particularly, trastuzumab,and inhibits downstream signaling through PcG-dependent epigeneticregulation of ErbB ligand sheddases ADAM10/17. Accordingly, it wasconfirmed that MEL-18 serves as a promising prognostic factor orpredictor for responses to anti-HER2 therapy in HER2-positive cancers,and when MEL-18 is used, resistance to HER2-targeted therapy can beovercome, and thus the present invention was completed.

Technical Solution

The present invention provides a composition for companion diagnosticsfor HER2-targeted drugs against HER2-positive cancers, the compositionincluding an agent for measuring the copy number of MEL-18 gene, themRNA expression level of the MEL-18 gene and/or the expression level ofMEL-18 protein in order to suggest a novel therapeutic strategy forHER2-positive cancers.

The composition for companion diagnostics may be for providinginformation on, specifically, the need for administration ofHER2-targeted drugs; the need for administration of ADAM10 and/or ADAM17inhibitor(s); the possibility of manifesting drug resistance toHER2-targeted drugs; the sensitivity to HER2-targeted drugs; predictionof prognosis after treatment with HER2-targeted drugs; or the need forco-administration of HER2-targeted drugs and ADAM10 and/or ADAM17inhibitor(s).

The cancer may be, but is not limited to, selected from the groupconsisting of breast cancer, gastric cancer, lung cancer, esophagealcarcinoma, bladder cancer and colon cancer.

In the composition, an agent for measuring the copy number of MEL-18gene or the mRNA expression level of the MEL-18 gene may be selectedfrom a sense primer, an antisense primer and a probe, whichcomplementarily bind to the MEL-18 gene or mRNA thereof.

In the composition, an agent for measuring a protein level may beselected from the group consisting of an antibody, an aptamer and aprobe, which specifically bind to MEL-18 protein.

The HER2-targeted drug may be any drug targeting HER2, and may be, butis not limited to, selected from the group consisting of trastuzumab,pertuzumab and trastuzumab emtansine (T-DM1).

In addition, the present invention provides a companion diagnosticmethod for HER2-targeted drugs against HER2-positive cancers, the methodincluding measuring the copy number of MEL-18 gene; the mRNA expressionlevel of the MEL-18 gene; or the expression level of MEL-18 protein in asample obtained from subjects with HER2-positive cancers.

The companion diagnostic method may be for providing information on theneed for administration of HER2-targeted drugs; the need foradministration of ADAM10 and/or ADAM17 inhibitor(s); the possibility ofmanifesting drug resistance to HER2-targeted drugs; the sensitivity toHER2-targeted drugs; prediction of prognosis after treatment withHER2-targeted drugs; or the need for co-administration of HER2-targeteddrugs and ADAM10 and/or ADAM17 inhibitor(s).

The companion diagnostic method may further include, when the copynumber of the MEL-18 gene; the mRNA expression level of the MEL-18 gene;or the expression level of the MEL-18 protein in a sample is lower thana predetermined value, providing information on the possibility thatsubjects can fall under one or more of the following categories,determined from the measurement result:

the need for administration of ADAM10 and/or ADAM17 inhibitor(s);

a high possibility of manifesting drug resistance to HER2-targeteddrugs;

low sensitivity to HER2-targeted drugs;

a poor prognosis after treatment with HER2-targeted drugs; or

the need for co-administration of HER2-targeted drugs with ADAM10 and/orADAM17 inhibitor(s).

The companion diagnostic method may further include providinginformation on one or more selected from the group consisting of MEL-18gene; MEL-18 gene expression activators; MEL-18 proteins; MEL-18 proteinexpression activators; ADAM10 gene inhibitors; ADAM17 gene inhibitors;ADAM10 protein expression inhibitors; ADAM17 protein expressioninhibitors; ADAM10 protein activity inhibitors; and ADAM17 proteinactivity inhibitors, or providing one or more thereof with HER2-targeteddrugs in order to treat HER2-positive cancers of the individual.

The HER2-targeted drug may be any drug targeting HER2, and may be, butis not limited to, selected from the group consisting of trastuzumab,pertuzumab and T-DM1.

In this method, the gene copy number; the mRNA expression level of theMEL-18 gene; or the expression level of the MEL-18 protein may beverified by a method selected from the group consisting of fluorescentin situ hybridization (FISH), comparative genomic hybridization(CGH-based array), a single nucleotide polymorphism (SNP) array,sequence assembly comparison, paired-end sequencing, multiplex ligationdependent probe amplification (MLPA), multiplex amplifiable probehybridization (MAPH), quantitative multiplex PCR of short fluorescentfragments (QMPSF), microsatellite genotyping, Southern blotting,immunohistochemistry polymerase chain reaction (PCR), quantitative PCR(qPCR), quantitative real-time PCR (qRT-PCR), real-time PCR,microarray-based comparative genomic hybridization and ligase chainreaction (LCR).

In addition, the present invention provides a kit for companiondiagnostics for HER2-targeted drugs, which includes an agent formeasuring the copy number of MEL-18 gene; a MEL-18 mRNA expressionlevel; and/or a MEL-18 protein expression level.

The kit may further include an agent for measuring the copy number ofthe HER2 gene, the mRNA expression level of the HER2 gene or theexpression level of the HER2 protein.

In the kit, the agent for measuring the copy number of a gene or themRNA expression level of the gene may be selected from the groupconsisting of a sense primer, an antisense primer and a probe, whichcomplementarily bind to the MEL-18 gene or mRNA thereof or the HER2 geneor mRNA thereof.

In the kit, the agent for measuring a protein expression level may beselected from the group consisting of an antibody, an aptamer and aprobe, which specifically bind to the MEL-18 protein or the HER2protein.

The kit may be for providing information on the need for administrationof HER2-targeted drugs; the need for administration of ADAM10 and/orADAM17 inhibitor(s); the possibility of manifesting drug resistance toHER2-targeted drugs; the sensitivity to HER2-targeted drugs; predictionof prognosis after treatment with HER2-targeted drugs; or the need forco-administration of HER2-targeted drugs and ADAM10 and/or ADAM17inhibitor(s).

In addition, the present invention provides an anticancer pharmaceuticalcomposition, which includes one or more selected from the groupconsisting of MEL-18 genes, MEL-18 gene expression activators, MEL-18proteins, MEL-18 protein activators, ADAM10 gene inhibitors, ADAM10protein activity inhibitors, ADAM17 gene inhibitors, and ADAM17 proteinactivity inhibitors as an active ingredient, for treating subjects withHER2-positive cancers that have the copy number of MEL-18 gene, the mRNAexpression level of the MEL-18 gene, and/or the expression level ofMEL-18 protein, which are/is lower than predetermined value(s); and/orhave or expected to have drug resistance to HER2-targeted drugs.

In addition, the present invention provides a pharmaceuticalcomposition, which includes one or more selected from the groupconsisting of MEL-18 gene, MEL-18 gene expression activators, MEL-18proteins, MEL-18 protein activators, ADAM10 gene inhibitors, ADAM10protein activity inhibitors, ADAM17 gene inhibitors, and ADAM17 proteinactivity inhibitors as an active ingredient, for inhibiting or improvingresistance to HER2-targeted drugs in HER2-positive cancer patients.

In addition, the present invention provides a pharmaceuticalcomposition, which includes one or more selected from the groupconsisting of MEL-18 gene, MEL-18 gene expression activator, MEL-18protein, the MEL-18 protein activator, ADAM10 gene inhibitors, ADAM10protein activity inhibitor, ADAM17 gene inhibitor, and ADAM17 proteinactivity inhibitor as an active ingredient, for assisting treatment of aHER2+ cancer patient with HER2-targeted drugs.

In the pharmaceutical composition, the ADAM10 and/or ADAM17 geneinhibitor(s) may be one or more selected from the group consisting of anantisense nucleotide, small interfering RNA (siRNA) and short hairpinRNA (shRNA), which complementarily bind to mRNA of ADAM10 and/or ADAM17gene(s).

In the composition, the ADAM10 and/or ADAM17 protein activityinhibitor(s) may be one or more selected from the group consisting of acompound, a peptide, a peptide mimetic, an aptamer, an antibody, and anatural substance, which specifically bind to ADAM10 and/or ADAM17protein(s).

In the composition, the ADAM10 and/or ADAM17 protein activityinhibitor(s) may be one or more selected from the group consisting ofINCB3619, INCB7839, WAY-022, TMI-2, CGS 27023, GW280264 and GI254023.

The composition may be administered together with HER2-targeted drugs,or simultaneously or sequentially co-administered with HER2-targeteddrugs.

The HER2-targeted drug may be any drug targeting HER2, and may be, butis not limited to, selected from the group consisting of trastuzumab,pertuzumab and T-DM1.

In addition, the present invention provides a composition for screeningan agent that improves resistance to HER2-targeted drugs or isco-administered with HER2-targeted drugs, which includes one or moreselected from the group consisting of MEL-18 gene, MEL-18 protein,ADAM10 gene, ADAM10 protein, ADAM17 gene and ADAM17 protein.

In addition, the present invention provides a method of screening a drugthat improves resistance to HER2-targeted drugs or is co-administeredwith HER2-targeted drugs, which includes: contacting candidate materialswith one or more selected from the group consisting of MEL-18 gene,MEL-18 protein, ADAM10 gene, ADAM10 protein, ADAM17 gene and ADAM17protein; and selecting a candidate material that increases the copynumber of the MEL-18 gene, the mRNA expression level of the MEL-18 gene,or the expression level of a protein expressed from the gene, orinhibits the expression level(s) of the ADAM10 and/or ADAM17 gene(s) orthe expression level of a protein expressed from the gene.

The screening method may further include judging a candidate materialthat promotes amplification of the MEL-18 gene; the mRNA expressionlevel of the MEL-18 gene; or the expression level of a protein expressedfrom the gene or inhibits the mRNA expression level(s) of the ADAM10and/or ADAM17 gene(s) or the expression level of a protein expressedfrom the gene to be a drug for inhibiting or improving resistance toHER2-targeted drugs.

In addition, the present invention provides a method of treatingsubjects having HER2-positive cancers, which includes administering apharmaceutical composition including one or more selected from the groupconsisting of MEL-18 gene; MEL-18 gene expression activators; MEL-18proteins; MEL-18 protein expression activators; ADAM10 gene inhibitors;ADAM17 gene inhibitors; ADAM10 protein expression inhibitors; ADAM17protein expression inhibitors; ADAM10 protein activity inhibitors; andADAM17 protein activity inhibitors to the subjects with HER2-positivecancers, wherein subjects have the copy number of MEL-18 gene, the mRNAexpression level of the MEL-18 gene, and/or the expression level ofMEL-18 protein, which are/is lower than predetermined value(s); and/orsubjects have or expected to have drug resistance to HER2-targeteddrugs.

The method may further include co-administering HER2-targeted drugs tosubjects receiving administration of the pharmaceutical composition.

Before administration of the pharmaceutical composition, the method mayfurther include measuring the copy number of the MEL-18 gene, the mRNAexpression level of the MEL-18 gene, or the expression level of theMEL-18 protein from a sample obtained from the individual withHER2-positive cancers.

In this method, the ADAM10 and/or ADAM17 gene or its mRNA expressioninhibitor may be one or more selected from the group consisting of anantisense nucleotide, small interfering RNA (siRNA) and short hairpinRNA (shRNA), which complementarily bind to mRNA of ADAM10 and/or ADAM17gene(s).

In this method, the ADAM10 and/or ADAM17 protein activity inhibitor maybe one or more selected from the group consisting of a compound, apeptide, a peptide mimetic, an aptamer, an antibody, and a naturalsubstance, which specifically bind to ADAM10 and/or ADAM17 protein(s).

In this method, the ADAM10 and/or ADAM17 protein activity inhibitor(s)may be one or more selected from the group consisting of INCB3619,INCB7839, WAY-022, TMI-2, CGS 27023, GW280264 and GI254023.

In addition, the present invention provides a use of an agent formeasuring the copy number of MEL-18 gene; the mRNA expression level ofthe MEL-18 gene; and/or the expression level of MEL-18 protein in asample obtained from subjects with HER2-positive cancers in companiondiagnostics for HER2-targeted drugs.

In addition, the present invention provides a use of an agent formeasuring the copy number of MEL-18 gene; the mRNA expression level ofthe MEL-18 gene; and/or the expression level of MEL-18 protein toprepare a composition for identifying subjects with HER2-positivecancers, wherein subjects have the copy number of MEL-18 gene, the mRNAexpression level of the MEL-18 gene, and/or the expression level ofMEL-18 protein, which are/is lower than predetermined value(s); and/orsubjects have or expected to have drug resistance to HER2-targeteddrugs.

The present invention also provides a use of an agent for measuring thecopy number of MEL-18 gene; the mRNA expression level of the MEL-18gene; and/or the expression level of MEL-18 protein to prepare acomposition for companion diagnostics for HER2-targeted drugs withrespect to subjects with HER2-positive cancers, wherein subjects havethe copy number of MEL-18 gene, the mRNA expression level of the MEL-18gene, and/or the expression level of MEL-18 protein, which are/is lowerthan predetermined value(s); and/or subjects have or expected to havedrug resistance to HER2-targeted drugs.

In addition, the present invention provides a use of an agent formeasuring the copy number of MEL-18 gene; the mRNA expression level ofthe MEL-18 gene; or the expression level of MEL-18 protein in a sampleobtained from subjects with HER2-positive cancers to prepare a kit forcompanion diagnostics for HER2-targeted drugs.

In addition, the present invention provides an application of one ormore selected from the group consisting of MEL-18 gene, MEL-18 geneexpression activator, MEL-18 protein, the MEL-18 protein activator,ADAM10 gene inhibitors, ADAM10 protein activity inhibitors, ADAM17 geneinhibitors and ADAM17 protein activity inhibitors to prepare a drug fortreating subjects with HER2-positive cancers, wherein subjects have thecopy number of MEL-18 gene, the mRNA expression level of the MEL-18gene, and/or the expression level of MEL-18 protein, which are/is lowerthan predetermined value(s); and/or subjects have or expected to havedrug resistance to HER2-targeted drugs.

Advantageous Effects

MEL-18 of the present invention is a prognostic factor and a predictorfor a response of subjects to an anti-HER2-targeted drug againstHER2-positive cancers, and can be used in companion diagnostics forHER2-targeted drugs for subjects with HER2-positive cancers, therebydetermining whether an ADAM10 or 17 inhibitor should be administered orshould be co-administered with HER2-targeted drugs, and thus overcomingresistance to the anti-HER2 therapy and improving its therapeuticefficacy. Therefore, HER2-positive cancers can be more effectivelytreated.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A shows that MEL-18 is associated with a positive survival resultin HER2+ breast cancer, which represents the percentage of cases withgenetic variation of MEL-18 in different breast cancer cohorts (TCGA andMETABRIC; left) and a MEL-18 gene amplification status from a HYUcohort, determined by FISH analysis (right). In the lower right, thereis the representative image of the case with MEL-18 amplification(MEL-18 signal: red; CEP17: green; DAPI: blue. MEL-18amp represents aMEL-18-amplified case; and MEL-18 non-amp represents a MEL-18non-amplified case).

FIG. 1B shows that MEL-18 is associated with positive survival resultsin HER2+ breast cancer, and scatterplots showing the correlation betweenMEL-18 expression and amplification in the indicated datasets. The rvalue was calculated through Pearson's correlation coefficient analysis.

FIG. 1C shows that MEL-18 is associated with positive survival resultsin HER2+ breast cancer, and scatterplots showing the correlation betweenMEL-18 expression and HER2 expression (top) and boxplots of MEL-18expression levels in different breast cancer subtypes from the indicateddatasets (bottom). Based on one-way ANOVA, ***P<0.001.

FIG. 1D shows that MEL-18 is associated with positive survival resultsin HER2+ breast cancer, and graphs showing OS and DFS analysis resultsrelative to MEL-18 amplification among 277 HER2-amplified breast cancercases from METABRIC. Data was analyzed using the Kaplan-Meier methodwith the log-rank test.

FIG. 1E shows DFS (disease-free survival) analysis of breast cancercases treated with trastuzumab (left, clinical cohort, n=213; right,GSE55348, n=53) according to MEL-18 amplification (left) or mRNAexpression (right). GSE55348 (cohort from GEO) was divided into twogroups according to the 66th percentile of MEL-18 expression(high, >66%; low, <33%). Data was analyzed using the Kaplan-Meier method(left) or Gehan-Breslow-Wilcoxon test (right) with the log-rank test.

FIG. 2A shows the result confirming that MEL-18 inhibition inducestrastuzumab resistance in HER2+ breast cancer according to an exemplaryembodiment of the present invention, in which the data shows MEL-18amplification and expression in HER2-amplified breast cancer cell lines(left) and a scatterplot shows the correlation between MEL-18 expressionand amplification, obtained by the Pearson's correlation coefficientanalysis (right).

FIG. 2B shows the result confirming that MEL-18 inhibition inducestrastuzumab resistance in HER2+ breast cancer according to an exemplaryembodiment of the present invention, in which an image of the MEL-18amplification status measured by FISH in each type of breast cancer celllines (BT474, SKBR3) (left), and MEL-18 mRNA levels in respective breastcancer cell lines (BT474, MDA-MB-361, HCC1954 and SKBR3) (right; mean±SD(n=3), P<0.05 (Welch's ANOVA)) are shown.

FIG. 2C shows the effect of MEL-18 on trastuzumab resistance in HER2+breast cancer cell lines according to an exemplary embodiment of thepresent invention. Stable knockdown or overexpression of MEL-18 in eachcell line was confirmed by immunoblotting (top). Cell survival aftertreatment with trastuzumab (m/ml) or a vehicle (Con) for 5 days wasanalyzed using the SRB analysis (bottom). shCon represents a shRNAcontrol; shMEL represents MEL-18 shRNA; Con represents an empty vector;and MEL represents MEL-18.

FIG. 2D shows the result confirming that MEL-18 inhibition inducestrastuzumab resistance in HER2+ breast cancer according to an exemplaryembodiment of the present invention, and graphs showing cell survivalafter treatment with 10 μg/ml of trastuzumab (Trz) or a vehicle (mean±SD(n=3), ***P<0.001 control (Con)(one-way ANOVA)).

FIG. 2E shows the result confirming that MEL-18 inhibition inducestrastuzumab resistance in HER2+ breast cancer according to an exemplaryembodiment of the present invention, and tumor growth curves forNOD/SCID mice injected with a control or MEL-18 knockdown BT474 cells(Arrows indicate the time when treatment was started with trastuzumab ora vehicle; mean±SEM (n=7); P<0.05 relative to the control (Con orshCon); and ^(554 †)P<0.01 relative to the vehicle-treated control(one-way ANOVA)).

FIG. 2F shows the immunohistochemistry results for MEL-18 and Ki-67 inthe xenografted tumors from each group according to an exemplaryembodiment of the present invention (mean±SD, n=5). IRS, immunoreactivescore. The scale bar, 100 μm. *P<0.05 relative to the shCon and †P<0.05relative to the vehicle (one-way ANOVA and post-hoc LSD test).

FIG. 3A shows that MEL-18 regulates ErbB phosphorylation and downstreamsignals according to an exemplary embodiment of the present invention,and a heatmap image obtained from the microarray analysis showing thedifferential expression of RTK signal-associated markers in a control(shCon) and MEL-18 shRNA (shMEL).

FIG. 3B shows that MEL-18 regulates ErbB phosphorylation and downstreamsignals according to an exemplary embodiment of the present invention,showing that the correlation of the gene expression profile ofBT474/shMEL-18 cells with each gene set (top) and the result of geneontology (GO) analysis of MEL-18 target genes (bottom).

FIG. 3C shows that MEL-18 regulates ErbB phosphorylation and downstreamsignals according to an exemplary embodiment of the present invention,and the expression of each gene measured by qRT-PCR (data is expressedas mean±SD (n=3), and based on the two-tailed Student's t test,***P<0.001 vs. control (shCon or Con).

FIG. 3D shows that MEL-18 regulates ErbB phosphorylation and downstreamsignals according to an exemplary embodiment of the present invention,showing phosphorylation-RTK analysis results for SKBR3 cells expressingMEL-18 or the control representing different phosphorylation levels ofRTK (spot analysis, top) and changes in relative band intensity measuredusing the AlphaEaseFC Software (bar graph, bottom).

FIG. 3E shows that MEL-18 regulates ErbB phosphorylation and downstreamsignals according to an exemplary embodiment of the present invention,and shows immunoblotting results for MEL-18-knockdown (shMEL) orMEL-18-overexpressing (MEL) breast cancer cells (BT474, ZR-75-30, SKBR3,HCC-1419) and control cells (shCon and Con), representing RTK anddownstream phosphorylation changes and relative expression.

FIG. 4A shows the results obtained from by ADAM10 and ADAM17downregulation by MEL-18 according to an exemplary embodiment of thepresent invention, and immunoblotting results showing changes in ADAM10and ADAM17 expression at the protein level due to MEL-18 knockdown oroverexpression. SKBR3 and HCC-1419 cell lines (MEL represents aMEL-18-overexpressing experimental group, and Con represents a control)dominantly expressed the mature form of ADAM10 (˜65 kDa), and BT474 andZR-75-30 cell lines (shMEL represents a MEL-18-knockdown experimentalgroup, and shCon represents a control) expressed a pro form of ADAM10(˜80 kDa).

FIG. 4B shows the results obtained from by ADAM10 and ADAM17downregulation by MEL-18 according to an exemplary embodiment of thepresent invention, and qRT-PCR results showing changes in mRNA levels ofADAM10 and ADAM17 in stable cell lines. In BT474 cells, shMEL representsa MEL-18-knockdown experimental group, shCon represents a control, andin SKBR3 cells, MEL represents a MEL-18-overexpressing experimentalgroup, and Con represents a control. The qRT-PCR results are expressedas mean±SD of triplicate measurements, and based on the two-tailedStudent's t test, *P<0.05 or ***P<0.001 relative to the control (shConor Con).

FIG. 4C shows the results obtained from by ADAM10 and ADAM17downregulation by MEL-18 according to an exemplary embodiment of thepresent invention, and ChIP-qPCR analysis results showing the amount ofproteins recruited to the promoter regions of ADAM10 and ADAM17 in aMEL-18-overexpressing (MEL) breast cancer cell line SKBR3 (top), aMEL-18-knockdown (shMEL) breast cancer cell line BT474 (bottom), andcontrol cells (shCon and Con). Data is expressed as mean±SD oftriplicate measurements, based on the two-tailed Student's t test,***P<0.001 relative to the control (shCon or Con), and IgG is a negativecontrol.

FIG. 4D shows the co-immunoprecipitation results for the binding levelsof MEL-18 with EZH2 and RING1B according to ADAM10 and ADAM17 downregulation by MEL-18 according to an exemplary embodiment of the presentinvention. After immunoprecipitation (IP) with an antibody againstMEL-18, immunoblotting (IB) was performed using the indicated antibody;a MEL-18-overexpressing (MEL) breast cancer cell line SKBR3 and controlcells (Con).

FIG. 4E shows a gene silencing model suggested by MEL-18 according to anexemplary embodiment of the present invention.

FIG. 5A shows that ADAM10/17 expression increased by MEL-18 knockdownincreases ErbB phosphorylation and induces trastuzumab resistance inHER2+ breast cancer according to an exemplary embodiment of the presentinvention, and the ELISA results confirming the protein levels of NRG1ligands present in the culture medium supernatant of SKBR3 cell lines(MEL is a MEL-18-overexpressing experimental group, and Con is acontrol) or BT474 cell lines (shMEL is a MEL-18-knockdown experimentalgroup, and shCon is a control). Data is expressed as mean±SD oftriplicate measurements, based on the two-tailed Student's t test**P<0.01, ***P<0.001 relative to the control (shCon or Con).

FIG. 5B shows that ADAM10/17 expression increased by MEL-18 knockdownincreases ErbB phosphorylation and induces trastuzumab resistance inHER2+ breast cancer according to an exemplary embodiment of the presentinvention, and the results of immunoprecipitation and immunoblotting forSKBR3 cell lines (MEL is a MEL-18-overexpressing experimental group, andCon is a control) and BT474 cell lines (shMEL is a MEL-18-knockdownexperimental group, and shCon is a control), performed to confirm adimerization status between HER2 and EGFR.

FIG. 5C shows that ADAM10/17 expression increased by MEL-18 knockdownincreases ErbB phosphorylation and induces trastuzumab resistance inHER2+ breast cancer according to an exemplary embodiment of the presentinvention, and changes in the expression levels and phosphorylation ofErbB family proteins in lysates of BT474 cell lines obtained bytreatment with 1 μM GW280264 (GW), which is an ADAM10/17 inhibitor, for24 hours, measured by immunoblotting (shMEL is a MEL-18-knockdownexperimental group, and shCon is a control).

FIG. 5D shows that ADAM10/17 expression increased by MEL-18 knockdownincreases ErbB phosphorylation and induces trastuzumab resistance inHER2+ breast cancer according to an exemplary embodiment of the presentinvention, and changes in the secretion levels of ErbB ligands aftertreatment of BT474 cell lines with GW280264 (GW) (shMEL is aMEL-18-knockdown experimental group, shCon is a control, and vehicle isa control).

FIG. 5E shows that ADAM10/17 expression increased by MEL-18 knockdownincreases ErbB phosphorylation and induces trastuzumab resistance inHER2+ breast cancer according to an exemplary embodiment of the presentinvention, and cell viability after treatment of BT474 cell lines withtrastuzumab (Trz) and/or GW280264 (GW) or a vehicle for 5 days,confirmed by an SRB assay (top, graph) and a colony-forming assay(bottom, image). The SRB assay data is expressed as mean±SD (n=3),**P<0.001 vs. a control (shCon), ^(†††)P<0.001 vs. a vehicle-treatedgroup and ^(‡)P<0.001 were based on one-way ANOVA (shMEL is aMEL-18-knockdown experimental group, shCon is a control, vehicle is acontrol).

FIG. 5F shows that ADAM10/17 mediates MEL-18 loss-induced ErbBphosphorylation and trastuzumab resistance, and the results ofimmunohistochemical analysis for ADAM10 and ADAM17 expression in tumortissues obtained from xenografted mice of each group. IRS is animmunoreactive score. The scale bar is 100 μm. Mean±SD (n=5). *P<0.05vs. shCon (Student's t-test) (shMEL is a MEL-18-knockdown experimentalgroup, and shCon is a control).

FIG. 5G shows the results of survival analysis of breast cancer patientstreated with trastuzumab according to the amplification status (left) orexpression levels (right) of MEL-18 in combination with ADAM10/17expression (A, amplification; NA, non-amplification; H, high; L, low).In a cohort from public data (GSE55348, n=53), patients were dividedinto four groups by various quantile expressions of indicated genes(cutoffs of high expression group: MEL-18, 66th percentile; ADAM10: 25thpercentile; ADAM17: 33th percentile). Data was analyzed using theKaplan-Meier method with the log-rank test (left) or theGehan-Breslow-Wilcoxon test (right).

FIG. 6 shows a model suggested to regulate ErbB signaling andtrastuzumab responses by MEL-18 in HER2+ breast cancer according to anexemplary embodiment of the present invention.

FIG. 7A shows MEL-18 amplification patterns in various types of humancancer according to an exemplary embodiment of the present invention,and graphs showing the percentage of MEL-18 amplification in HER2amplification cases of various cancer type public datasets. Here, amprepresents an amplified case; and non-amp represents a non-amplifiedcase.

FIG. 7B shows MEL-18 amplification patterns in various types of humancancer according to an exemplary embodiment of the present invention,and graphs showing the co-amplification percentage of different geneslocated in a 17q12-21 amplicon with HER2 gene and distances from theHER2 gene locus.

FIG. 8A shows the result of the functional analysis of MEL-18 targetgenes by a microarray using Gene Set Enrichment Analysis (GSEA)according to an exemplary embodiment of the present invention,indicating GSEA with genes differentially expressed fromMEL-18-knockdown BT474 cells, determined by a microarray analysis.

FIG. 8B shows the function of a MEL-18 target gene analyzed by amicroarray using GSEA according to an exemplary embodiment of thepresent invention, and plots of gene sets having the statisticallypositive correlation with MEL-18 knockdown derived from different canceroccurring pathways.

FIGS. 9A and 9B show the results of ChIP-seq analysis for MEL-18 bindingto promoter sites of ADAM family genes, and the example of a peak mapfor ChIP-seq data of MEL-18 targets from mouse embryonic stem cells(GSE67868).

MODES OF THE INVENTION

Hereinafter, the present invention will be described in detail.

However, the present invention can be modified in various ways, and havevarious forms, and thus specific embodiments and descriptions to beprovided below are merely to help in understanding of the presentinvention, but not to limit the present invention to specificembodiments. It should be understood that the scope of the presentinvention includes all modifications, equivalents and alternativeswithin the idea and technical scope of the present invention.

The present invention relates to a composition for companion diagnosticsfor HER2-targeted drugs against HER2-positive cancers, which includes anagent for measuring the copy number of MEL-18 gene, the mRNA expressionlevel of the MEL-18 gene and/or the expression level of MEL-18 protein.

MEL-18 is a protein of the polycomb group (PcG) belonging topolycomb-repressive complex 1 (PRC1). PcG proteins are a crucialepigenetic regulators consisting of two multicomplexes: one is PRC2,which is an initiation complex of a silencing gene, catalyzing histone 3tri-methylation in lysine-27 (H3K27me3); and the other is PRC1, whichrecognizes H3K27me3 and maintains gene silencing via various mechanismsincluding histone H2A mono-ubiquitination on lysine-119 (H2AK119ub) andblockade of transcriptional initiation machinery (1-3). MEL-18 is alsoknown as polycomb group ring finger 2, RNF110, or ZNF144, and NCBI orthe like may be referenced for its gene, mRNA and protein data. Forexample, it is registered under NCBI Gene ID: 454614 (Homo sapiens(human)); NCBI Reference Sequence: NP_009075.1; or NCBI Gene ID: 7703,and includes all sequence data known as MEL-18, as well as the genedata.

In an exemplary embodiment of the present invention, it was confirmedthat, when MEL-18 gene is not amplified or MEL-18 mRNA is less expressedin subjects with HER2+ breast cancer, MEL-18 has resistance totrastuzumab, which is HER2-targeted drugs, and when overexpression ofMEL-18 is induced or ADAM10 and/or ADAM17 inhibitor(s) is(are) treated,resistance to trastuzumab is improved. In addition, MEL-18 is amplifiedonly in half of individuals with HER2+ gastric cancer, lung cancer oresophageal carcinoma, similar to the breast cancer case. Accordingly,the MEL-18 of the present invention has a novel application as a markerfor companion diagnostics to determine whether HER2-targeted drugs, orADAM10 and/or ADAM17 inhibitor(s) should be applied, or appliedindividually or in combination to subjects with HER2-positive cancers.

The term “MEL-18” used herein is interpreted to include all of MEL-18gene, mRNA of the MEL-18 gene, and MEL-18 protein. In addition, theMEL-18, MEL-18 gene or MEL-18 protein is understood to include afragment or recombinant protein thereof having substantially the sameactivity as them, and codon-optimized cDNA. Accordingly, in the presentinvention, a MEL-18 expression level means the mRNA expression level ofthe MEL-18 gene or the expression level of the MEL-18 protein expressedfrom the gene. In addition, the term “HER2,” “ADAM10,” or “ADAM17” maybe interpreted as a meaning including all of a gene, mRNA and a proteinthereof.

The term “companion diagnostics” used herein means one of diagnostictests for verifying the possibility of applying a specific drug to aspecific patient, and in the present invention, to verify thepossibility of applying HER2-targeted drugs or ADAM10 and/or ADAM17inhibitor(s) to subjects with HER2-positive cancers, the copy number ofthe MEL-18 gene, its mRNA expression level or the expression level ofthe MEL-18 protein expressed from the gene may be measured, as acompanion diagnostic marker, in individuals with the above-mentionedcancer. Therefore, in this aspect, the companion diagnostics used hereinmay be performed to provide information on the need for administrationof HER2-targeted drugs; the need for administration of ADAM10 and/orADAM17 inhibitor(s); the possibility of manifesting drug resistance toHER2-targeted drugs; the sensitivity to HER2-targeted drugs; predictionof prognosis after treatment with HER2-targeted drugs; or the need forco-administration of HER2-targeted drugs and ADAM10 and/or ADAM17inhibitor(s).

The term “determination” used herein means distinguishing objectsaccording to specific criteria. In the present invention, determinationmay be used as a meaning of distinguishing whether subjects diagnosed tohave HER2-positive cancers or likely to have the cancer have or do nothave drug resistance to HER2-targeted drugs, or whether theabove-mentioned subjects have or do not have sensitivity toHER2-targeted drugs.

In addition, the expression “need for administration” is used herein forthe purpose of determining whether a specific agent (drug) should beadministered only to subjects expected to have a therapeutic effect withrespect to the agent (drug), and such determination may be made bymeasurement of the level of a companion diagnostic marker. Specifically,in the present invention, to determine whether to administerHER2-targeted drugs; ADAM10 and/or ADAM17 inhibitor(s); or a combinationof HER2-targeted drugs with ADAM10 and/or ADAM17 inhibitor(s), as acompanion diagnostic marker, the copy number of the MEL-18 gene, themRNA expression level thereof the expression level of the MEL-18 proteinexpressed from the gene may be measured.

The term “human epidermal growth factor receptor2 (HER2)-positivecancer” used herein means cancer exhibiting HER2 gene amplification oroverexpression, and may be, but is not limited to, selected from thegroup consisting of HER2+ breast cancer, gastric cancer, lung cancer,esophageal carcinoma, bladder cancer and colon cancer. In an exemplaryembodiment of the present invention, the HER2-positive cancers may beHER2+ breast cancer, HER2+ gastric cancer, or HER2+ lung cancer. Whetheror not the cancer is HER2-positive (exhibiting gene amplification oroverexpression) may be determined according to criteria generally usedin the art to which the present invention belongs, which may varyaccording to clinical criteria for diagnosing HER2 positive in eachcountry. In an exemplary embodiment of the present invention, it wasconfirmed that MEL-18 can be used as a companion diagnostic marker inHER2+ breast cancer cells, and in the cases of gastric cancer, lungcancer, esophageal carcinoma, bladder cancer and colon cancer, MEL-18amplification characteristics similar to breast cancer were observed. Itcan be seen that the MEL-18 of the present invention can be used as acompanion diagnostic marker for the above-mentioned types of cancer.

The composition for companion diagnostics according to the presentinvention includes an agent for measuring the copy number of the MEL-18gene; the mRNA expression level thereof; and/or the expression level ofthe MEL-18 protein.

The term “copy number of a gene” is the number of specific genes in aspecific genotype of each individual, meaning the number of a specificgene or gene fragment of which multiple copies are formed. It may beunderstood that the measurement of the copy number of a gene in thepresent invention is the same as measurement of a copy number variation(or copy number variant (CNV)) and a gene copy number (GCN), which aregenerally used in the art. In addition, when the copy number of a geneis same as or higher than a predetermined value, this can indicate thatthere is gene amplification, and when the copy number of a gene is thesame as or lower than the predetermined value, this can indicate thatthere is no gene amplification. In the present invention, the “geneamplification” can be used interchangeably with gene duplication, whichmeans that the number of gene copies is higher than the predeterminedvalue. In an exemplary embodiment of the present invention, among valuesobtained by measuring the copy number of a gene through FISH in subjectswith HER2+ breast cancer, when the copy number of the MEL-18 gene ishigher than 6.0, which is the predetermined value, it is considered thatthere is gene amplification, followed by performing analysis.

The term “mRNA expression level” or “protein expression level” means thedegree to which messenger RNA delivering the genetic information of aspecific gene to a ribosome or a protein is expressed from the specificgene. When the mRNA or protein expression level is higher than aparticular predetermined value (or a particular reference value), thisindicates “overexpression”. When the mRNA or protein expression level isthe same as or less than the predetermined value, this indicates “lowexpression”. In an exemplary embodiment of the present invention, aMEL-18 mRNA expression level (about 1 (TCGA in FIG. 1B), about 11(METABRIC in FIG. 1B); measured through RNA-seq or a microarray,z-scores) corresponding to MEL-18 CNA 2 or more was set as apredetermined value for analysis. In another exemplary embodiment of thepresent invention, among the measured values obtained from a microarray,a group in which the mRNA expression value of MEL-18 corresponding tothe top ⅔ (66%) was considered overexpressed (positive), followed byperforming analysis.

The term “agent for measuring the copy number of a gene” or “agent formeasuring an mRNA expression level” may be selected from the groupconsisting of a sense primer, an antisense primer and a probe, whichcomplementarily bind to a gene or mRNA thereof, but the presentinvention is not limited thereto.

The “primer” used herein refers to a nucleic acid sequence having ashort free 3′ hydroxyl group, and a short nucleic acid sequence whichcan form a complementary template and base pair and serving as astarting point for the replication of a template strand. The primer mayinitiate DNA synthesis in a suitable buffer solution at a suitabletemperature in the presence of a reagent for a polymerization reaction(DNA polymerase or reverse transcriptase) and four different dNTPs(deoxynucleoside triphosphates). In the present invention, the primer isa primer capable of specifically binding to the MEL-18 gene, preferablyconsisting of sense (forward) and antisense (reverse) nucleic acidshaving 7 to 50 nucleotide sequences. The primer may have an additionalcharacteristic that does not change the basic property of the primerserving as the starting point of DNA synthesis. In addition, the primernucleic acid sequence of the present invention may include a directly orindirectly detectable marker by spectroscopic, photochemical,biochemical, immunochemical or chemical means when needed.

In an exemplary embodiment of the present invention, the primer may besense and/or antisense primer(s) complementary to the MEL-18 gene or itsmRNA. The sense and antisense primers may be included alone or incombination. In addition, the primer may be a probe capable ofspecifically binding to MEL-18.

The “probe” used herein refers to a nucleic acid fragment such as RNA orDNA having a length of several nucleotides to several hundred bases,which can specifically bind to a specific nucleotide sequence. Whenlabeled, the primer includes a form of an oligonucleotide probe, asingle-stranded DNA probe, a double-stranded DNA probe or an RNA probe,which can confirm the presence or absence of a specific gene or itsmRNA.

The primer or probe of the present invention may be chemicallysynthesized using a known method.

The “method of measuring the gene copy number” used herein may use allof conventional methods used in the art to which the present inventionbelongs. More specifically, both of full-length genome analysis and atarget-specific method may be used. Specifically, a method selected fromthe group consisting of fluorescent in situ hybridization (FISH),comparative genomic hybridization (CGH-based array), a SNP array,sequence assembly comparison, paired-end sequencing, multiplex ligationdependent probe amplification (MLPA), multiplex amplifiable probehybridization (MAPH), quantitative multiplex PCR of short fluorescentfragments (QMPSF), microsatellite genotyping, Southern blotting,immunohistochemistry (IHC), polymerase chain reaction (PCR),quantitative PCR (qPCR), quantitative real-time PCR (qRT-PCR), real-timePCR, microarray-based comparative genome hybridization and ligase chainreaction (LCR) may be used, but the present invention is not limitedthereto.

The “method of measuring an mRNA expression level” used herein may useall of conventional methods used in the art to which the presentinvention belongs. More specifically, the method includes PCR, RT-PCR,competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA),Northern blotting, RNA-sequencing (RNA-seq), a nano string or a DNAchip, but the present invention is not limited thereto. The detectionmethods may be used to confirm overexpression/low expression of mRNA bycomparing the mRNA expression level of a biological sample obtained fromsubjects requiring companion diagnostics with the mRNA expression levelin a comparative sample, to confirm overexpression/low expression bymeasuring the mRNA expression levels of the samples and comparing themwith the predetermined value, or to confirm an increase/decrease in theexpression in a comparison in which the MEL-18 mRNA expression levelafter treatment with HER2-targeted drugs is lower than that beforetreatment with the HER2 agent.

The “agent for measuring a protein level” used herein may be selectedfrom the group consisting of an antibody, an aptamer and a probe, whichspecifically bind to a protein, but the present invention is not limitedthereto. In the present invention, the agent may be selected from thegroup consisting of an antibody, an aptamer and a probe, whichspecifically bind to the MEL-18 protein.

In addition, the “method of measuring a protein expression level” mayuse all of conventional methods used in the art to which the presentinvention belongs. More specifically, a protein chip analysis, animmunoassay, a ligand binding assay, Matrix Assisted LaserDesorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF),Surface-Enhanced Laser Desorption/Ionization Time of Flight MassSpectrometry (SELDI-TOF), a radioimmunoassay, a radioimmunoprecipitationassay, Ouchteroni immunodiffusion, rocket immunoelectrophoresis,immunohistochemistry (IHC), a complement fixation assay, two-dimensionalelectrophoresis analysis, liquid chromatography-mass spectrometry(LC-MS), liquid chromatography-mass spectrometry/mass spectrometry(LC-MS/MS), Western blotting or an enzyme linked immunosorbent assay(ELISA) may be used, but the present invention is not limited thereto.

The term “drug resistance” used herein means that there is nostatistically significant cellular or biological response to a specificdrug. Specifically, it means that the death rate of cancer cells isreduced or there is no apoptosis of the cancer cells due to treatmentwith a drug.

The term “HER2-targeted drug(s)” are/is used in combination with a “HER2gene or protein inhibitor,” and has an effect of inhibiting or hinderingthe expression or activity of the HER2 gene or protein inhibitor byspecifically acting on the HER2 gene or protein, and includes all typesof anti-HER2 agents. Specifically, it may be an expression or activityinhibitor through the inhibition of HER2 dimerization.

The HER2 expression inhibitor may be one or more selected from the groupconsisting of an antisense nucleotide, small interfering RNA (siRNA) andshort hairpin RNA (shRNA), which complementarily bind to mRNA of thegene.

The HER2 protein activity inhibitor may be one or more selected from thegroup consisting of a natural or synthetic compound, a peptide, apeptide mimetic, an aptamer and an antibody, which specifically bind tothe protein.

The HER2-targeted drug means a therapeutic agent targeting a HER2receptor, for example, an antibody therapeutic. In the presentinvention, as a HER2-targeted drug, any therapeutic targeting a HER2receptor, which has already been disclosed in papers and patents, otherthan medications which are now commercially available, may be included.Specifically, it has been identified that, in the present invention,signaling of a HER2 receptor is mediated by MEL-18, and it can be seenthat MEL-18-mediated companion diagnostics of determiningresistance/sensitivity to HER2-targeted drugs, restoring sensitivity ordetermining treatment with an ADAM10/17 inhibitor may be widely appliedregardless of the type of therapeutic agent, and therefore, alltherapeutic agents targeting a HER2 receptor may be included in thepresent invention. Specifically, the therapeutic agent targeting a HER2receptor may be selected from the group consisting of trastuzumab,pertuzumab and trastuzumab emtansine (T-DMI)), but the present inventionis not limited thereto.

The trastuzumab, also called Herceptin™, is a therapeutic agent formetastatic cancer which is triggered by overexpression of the HER2 geneor its gene product, and an antibody therapeutic agent recognizing anextracellular moiety of HER2 as an antigen epitope.

Pertuzumab, an anti-HER2 therapeutic agent for a metastatic disease, ischemically available under the trade name Perjeta. Pertuzumab blocksHER2 signaling to inhibit HER2 dimerization. Pertuzumab may be used incombination with trastuzumab or docetaxel.

Trastuzumab emtansine (T-DM1), also called ado-trastuzumab emtansine, isan antibody-drug conjugate which is commercially available under thetrade name Kadcyla. T-DM1 has a structure in which trastuzumab is linkedto the cytotoxic agent emtansine (DM1).

The term “individual” or “patient” used herein may be a vertebrate, andparticularly a mammal, and the mammal includes all of a dog, a horse, acat, a cow, a primate, a mouse and a rat, and preferably, a human.

In addition, the present invention relates to a method for companiondiagnostics for HER2-targeted drugs against HER2-positive cancers, whichincludes measuring the copy number of MEL-18 gene; the mRNA expressionlevel of the MEL-18 gene; or the expression level of MEL-18 protein insamples obtained from individuals with HER2-positive cancers.

All descriptions regarding the composition for companion diagnostics mayalso be applied or applied mutatis mutandis to the method for companiondiagnostics of the present invention.

The method for companion diagnostics may be for providing informationon, specifically, the need for administration of HER2-targeted drugs;the need for administration of ADAM10 and/or ADAM17 inhibitor(s); thepossibility of manifesting drug resistance to HER2-targeted drugs; thesensitivity to HER2-targeted drugs; prediction of prognosis aftertreatment with HER2-targeted drugs; or the need for co-administration ofHER2-targeted drugs and ADAM10 and/or ADAM17 inhibitor(s).

The method for companion diagnostics may further include, when the copynumber of the MEL-18 gene; the mRNA expression level of the MEL-18 gene;or the expression level of the MEL-18 protein in a sample is lower thana predetermined value, providing information on the possibility thatsubjects can fall under one or more of the following categories,determined from the measurement result:

the need for administration of ADAM10 and/or ADAM17 inhibitor(s);

a high possibility of manifesting drug resistance to HER2-targeteddrugs;

low sensitivity to HER2-targeted drugs;

a poor prognosis after treatment with HER2-targeted drugs; or

the need for co-administration of HER2-targeted drugs with ADAM10 and/orADAM17 inhibitor(s).

When the copy number of the gene and/or the mRNA expression level is thesame or less than the predetermined value, or the protein activity levelis the same or less than the predetermined value, it may be determinedas non-amplification (or negative) of the MEL-18 gene or low MEL-18expression, indicating no amplification or reduced expression of theMEL-18 gene.

An individual considered having non-amplification (negative) or lowexpression of MEL-18 through the measurement, that is, subjectsexhibiting the copy number of the MEL-18 gene; the mRNA expression levelof the MEL-18 gene; or the expression level of a protein encoded by theMEL-18 gene, which are lower than the predetermined values, may beclassified as one having low sensitivity to HER2-targeted drugs, or onehaving resistance to the therapeutic agent. In addition, when theindividual is one receiving administration of HER2-targeted drugs, theprognosis of treatment with HER2-targeted drugs may be predicted to bepoor, and the individual is considered to have drug resistance to theadministered HER2-targeted drug. Therefore, to increase sensitivity orreduce resistance to the HER2-targeted drug in an individual, it may bejudged that there is a need for administration (or prescription) of anADAM10 and/or ADAM17 inhibitor or co-administration of the HER2-targeteddrug with an ADAM10 and/or ADAM17 inhibitor(s); or administration (orprescription) of the MEL-18 gene, MEL-18 gene expression activators, theMEL-18 protein or a MEL-18 protein activator.

The term “predetermined value (or reference value)” means a benchmarkvalue for dividing amplification/non-amplification of a gene; oroverexpression/low expression of mRNA or a protein. The reference valuemay be, for example, an average gene copy number or an averagemRNA/protein expression level of subjects before treatment with aspecific drug, or an average gene copy number or an average mRNA/proteinexpression level of a normal individual, but the present invention isnot limited thereto. In addition, the reference value may be determinedaccording to the distribution of the average gene copy number or thedistribution of a mRNA/protein expression level of a specific patientgroup, but the present invention is not limited thereto.

Specifically, the gene copy number, the mRNA expression level or proteinexpression level is determined by measuring an average gene copy number,the mRNA expression level or protein expression level from a sample ofeach individual using a method of measuring a gene, mRNA or proteinlevel, which is conventionally used in the art of the present invention,and a measured value in the distribution of measured valuescorresponding to the top 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% may be set as a predetermined value.

For example, in the case of a MEL-18 gene copy number, the predeterminedvalue may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies or more.

In an exemplary embodiment of the present invention, a gene copy numberof each individual was measured by FISH, and the gene copy numberaccounting for the approximate top 50% was set as the benchmark fordividing positive/negative of the MEL-18 gene. Here, the predeterminedvalue was 6 copies, and when the gene copy number was more than 6.0copies (gene/chromosome ratio >6.0), it was classified as amplificationof the MEL-18 gene.

In another exemplary embodiment, copy number variation (CNV) wasdetected through whole-exome sequencing or the SNP array 6.0, and theresults were classified as follows using the GISTIC algorithm: (+2(amplification), +1 (duplication), 0 (neutral), −1 (deletion), −2(homozygous deletion), +2 or more (in the case of a gene copy number of4 or more, it was classified as amplification).

In another exemplary embodiment, a protein expression level is measuredby immunohistochemistry using a MEL-18 antibody, and the intensity andarea of staining were quantified, thereby obtaining the measured valueof protein expression. The final measured value (score) for the proteinexpression is obtained by multiplying two scores obtained by grading theprotein expression as follows: 0 (no cells stained), 1 (1% to 24% cellsstained), 2 (25% to 49% cells stained), 3 (50% to 74% cells stained),and 4 (75% to 100% cells stained) according to the percentage of theimmunostained cells, and 0 (no staining), 1 (weak staining), 2 (moderatestaining) and 3 (strong staining) according to the intensity of thestaining. The case in which the distribution of the final measuredvalues corresponds to the top 25% was set as the benchmark for dividingpositive/negative (or overexpression/low expression), and at this time,the measured value was 4. As a result, for samples or individuals withthe measured value of 4 or more, the protein expression may beconsidered overexpressed or positive.

In still another exemplary embodiment, analysis was carried out with theMEL-18 mRNA expression levels (about 1 (TCGA of FIG. 1B), about 11(METABRIC of FIG. 1B); measured through RNA-seq or microarray,z-scores), corresponding to MEL-18 CNA 2 or more, which were set aspredetermined values, and in yet another exemplary embodiment, from themeasured values obtained through a microarray, a group with MEL-18 mRNAexpression levels corresponding to top ⅔ (66%) was determined asoverexpressed (positive), followed by performing analysis.

Other than these, for the readout of the MEL-18 gene or protein, or thesetting of predetermined values, [Jeong-Yeon Lee et al., jci.org Volume125, Number 5, May 2015, “MEL-18 loss mediates estrogen receptor-αdownregulation and hormone independence”] may be referenced.

As described above, the predetermined value of the present invention maybe set according to a conventional method used in the art to which thepresent invention belongs, and may use various types of statisticalmethods and software for obtaining statistics. In the present invention,the predetermined value may vary according to the total number (n) ofsamples (or individuals) or a purpose of setting a predetermined value.While, when a kit is manufactured using the MEL-18 marker of the presentinvention, the predetermined value may vary according to an optimizationextent of the product, the predetermined value may be set with referenceto the present invention, and thus all of them are included in thepresent invention. In addition, the predetermined value of the presentinvention may be a predetermined value set by a conventional method.

In addition, the method may further include prescribing (or providinginformation for prescription) one or more selected from the groupconsisting of the MEL-18 gene, MEL-18 gene expression activators, theMEL-18 proteins, MEL-18 protein activators, ADAM10 gene inhibitors,ADAM10 protein activity inhibitors, ADAM17 gene inhibitors and ADAM17protein activity inhibitors to subjects classified as exhibiting no (ornegative) amplification of the MEL-18 gene or low MEL-18 expression. Inaddition, the method may further include prescribing (or providinginformation for prescription) one or more selected from the groupconsisting of the MEL-18 gene, MEL-18 gene expression activators, theMEL-18 proteins, MEL-18 protein activators, ADAM10 gene inhibitors,ADAM10 protein activity inhibitors, ADAM17 gene inhibitors and ADAM17protein activity inhibitors in combination with HER2-targeted drugs tosubjects classified as exhibiting no (or negative) amplification of theMEL-18 gene or low MEL-18 expression.

The term “sample” used herein may be urine, a body fluid (includingblood, lymph, or tissue fluid), or biopsy tissue, and preferably, oneobtained from subjects with cancer, and more preferably, one obtainedfrom subjects suspected of, determined or diagnosed with HER2-positivecancers.

The present invention also provides a kit or system for companiondiagnostics for HER2-targeted drugs, which includes an agent formeasuring the copy number of the MEL-18 gene; the mRNA expression levelof the MEL-18 gene; or the expression level of the MEL-18 protein.

All descriptions regarding the composition or method for companiondiagnostics may also be applied mutatis mutandis to the kit or systemfor companion diagnostics of the present invention.

The kit or system may further include an agent for measuring the copynumber of the HER2 gene; the mRNA expression level of the HER2 gene orthe expression level of the HER2 protein. In this case, the kit forcompanion diagnostics of the present invention may simultaneouslydetermine whether subjects who provided a sample is HER2+, and hasresistance (or sensitivity) to HER2-targeted drugs. Whether or not oneis HER2+ may be determined according to a method known in the art towhich the present invention belongs, and for example, in the case ofHER2+ breast cancer, it may be considered according to the criteriadisclosed in [J Clin Oncol. 2007 Jan. 1; 25(1):118-45. Epub 2006 Dec.11; PMID: 17159189], and when the HER2 gene copy number is more than 6,it may be considered positive. However, not limited to the aboveexamples, the criteria may vary depending on the type of HER2-positivecancers, and if there are methods and/or criteria decided by eachcountry according to clinical standards, whether or not one is HER2+ maybe determined thereby.

The agent for measuring the copy number or mRNA expression level of thegene may be selected from the group consisting of a sense primer, anantisense primer and a probe, which complementarily bind to each of theMEL-18 gene/mRNA or the HER2 gene/mRNA.

The agent for measuring a protein level may be selected from the groupconsisting of an antibody, an aptamer and a probe, which specificallybind to the MEL-18 protein or the HER2 protein.

The kit or system may be for providing information on, specifically, theneed for administration of HER2-targeted drugs; the need foradministration of ADAM10 and/or ADAM17 inhibitor(s); the possibility ofmanifesting drug resistance to HER2-targeted drugs; the sensitivity toHER2-targeted drugs; prediction of prognosis after treatment withHER2-targeted drugs; or the need for co-administration of HER2-targeteddrugs and ADAM10 and/or ADAM17 inhibitor(s).

In an exemplary embodiment, the need for prescription of an anti-HER2agent may be determined by verifying whether or not one is HER2+ from asample obtained from subjects with cancer, and measuring the copy numberof the MEL-18 gene, the mRNA expression level of the MEL-18 gene or aMEL-18 protein expression/activity level. Specifically, when theindividual has the copy number and/or mRNA expression level of theMEL-18 gene, which are lower than the predetermined values, theindividual may be defined as having low sensitivity to HER2-targeteddrugs, or having resistance to HER2-targeted drugs. In addition, whenthe individual receives the administration of HER2-targeted drugs, itmay be expected that the prognosis of treatment with HER2-targeted drugswill be poor, considering that the individual has drug resistance to theadministered HER2-targeted drug. Accordingly, to increase thesensitivity or reduce resistance to HER2-targeted drugs in theindividual, it may be considered that it is necessary to administerADAM10 and/or ADAM17 inhibitor(s), or co-administer ADAM10 and/or ADAM17inhibitor(s) with HER2-targeted drugs; or administer (or prescribe) theMEL-18 gene, MEL-18 gene expression activators, the MEL-18 protein or aMEL-18 protein activator.

The kit includes a diagnostic kit based on a conventional gene copynumber, and mRNA expression and protein quantitative analyses withoutlimitation. For example, the kit may be a RT-PCR kit, a DNA chip kit, aprotein kit or an array kit. For a RT-PCR kit, in addition to primerpairs specific to the MEL-18 gene and/or the HER2 gene, a test tube oranother suitable container, a reaction buffer, a deoxynucleotide (dNTP),enzymes such as Taq-polymerase and reverse transcriptase, a DNase orRNase inhibitor, and sterile water may be generally included ascomponents of the kit. In addition, conventional technology for a kit orsystem for companion diagnostics may be applied to the presentinvention.

In addition, the present invention relates to an anti-cancerpharmaceutical composition, which includes one or more selected from thegroup consisting of MEL-18 gene, MEL-18 gene expression activators,MEL-18 proteins, MEL-18 protein activators, ADAM10 gene inhibitors,ADAM10 protein activity inhibitors, ADAM17 gene inhibitors, and ADAM17protein activity inhibitors as an active ingredient, for treatingsubjects with HER2-positive cancers as well as having the copy number ofMEL-18 gene, the mRNA expression level of the MEL-18 gene, and/or theexpression level of MEL-18 protein, which are/is lower thanpredetermined value(s); and/or having or being expected to have drugresistance to HER2-targeted drugs.

In addition, the present invention relates to a pharmaceuticalcomposition, which includes one or more selected from the groupconsisting of MEL-18 gene, MEL-18 gene expression activators, MEL-18proteins, MEL-18 protein activators, ADAM10 gene inhibitors, ADAM10protein activity inhibitors, ADAM17 gene inhibitors, and ADAM17 proteinactivity inhibitors as an active ingredient, for inhibiting or improvingresistance to HER2-targeted drugs in subjects with HER2-positivecancers.

In addition, the present invention provides a pharmaceuticalcomposition, which includes one or more selected from the groupconsisting of MEL-18 gene, MEL-18 gene expression activators, MEL-18proteins, MEL-18 protein activators, ADAM10 gene inhibitors, ADAM10protein activity inhibitors, ADAM17 gene inhibitors, and ADAM17 proteinactivity inhibitors, for assisting treatment of HER2+ cancer patientswith HER2-targeted drugs.

All descriptions regarding the composition for companion diagnostics,the companion diagnostic method and the kit for companion diagnosticsmay also be applied mutatis mutandis to the pharmaceutical compositionof the present invention.

The MEL-18 gene may be included in the composition in the form of avector system including the MEL-18 gene, and the composition may beadministered in the form of a gene therapeutic agent.

The MEL-18 gene expression activator may also be a protein, compound ornucleic acid molecule, which can promote the expression of the MEL-18gene, or the nucleic acid molecule may be included in the composition inthe form of a vector system.

The MEL-18 protein may be administered by itself, or administered in theform of a vector system including an mRNA sequence capable of encodingthe protein. The MEL-18 protein activator may be a protein, compound ornucleic acid molecule which can promote the activity of the protein, andthe nucleic acid molecule may be included in the composition while beingincluded in a vector system in the form of a gene therapeutic agent. Theimplementation of the gene therapeutic agent or vector system may becarried out according to a method conventionally known in the technicalfield of the present invention.

In addition, the pharmaceutical composition of the present invention mayinclude one or more selected from the group consisting of ADAM10 geneinhibitors, ADAM10 protein activity inhibitors, ADAM17 gene inhibitors,and ADAM17 protein activity inhibitors. When subjects with HER2-positivecancers, wherein subjects have the copy number of MEL-18 gene, the mRNAexpression level of the MEL-18 gene, and/or the expression level ofMEL-18 protein, which are/is lower than predetermined value(s); and/orsubjects have or expected to have drug resistance to HER2-targeteddrugs, are treated with ADAM10 gene inhibitors, ADAM10 protein activityinhibitors, ADAM17 gene inhibitors and ADAM17 protein activityinhibitors, or co-treated with an ADAM10/17 inhibitor and HER2-targeteddrugs, resistance of the subject against HER2-targeted drugs may beinhibited, drug sensitivity and a therapeutic effect may be improved,and a prognosis may be improved. The ADAM10 gene and/or ADAM17 geneinhibitor inhibits the expression of ADAM10 and/or ADAM17 gene(s), andthe inhibition includes all of prevention of transcription by regulatinggene transcription, prevention of protein expression by inhibiting aprocess of translation from the mRNA of a gene to a protein, anddegradation of a protein and/or mRNA itself. Therefore, the geneinhibitor includes an inhibitor of the expression of the mRNA of a geneand an inhibitor of the expression of a protein encoded by the gene.

The inhibitor may be one or more selected from the group consisting ofan antisense nucleotide, microRNA (miRNA), small interfering RNA(siRNA), short hairpin RNA (shRNA), a compound and a protein, whichcomplementarily bind to ADAM10, ADAM17, or ADAM10 and ADAM17 genes.

The ADAM10 or ADAM17 protein activity inhibitor may be one or moreselected from the group consisting of a compound, a peptide, a peptidemimetic, an aptamer and an antibody, which specifically act on ADAM10,ADAM17, or ADAM10 and ADAM17 proteins. It is important that the presentinvention elucidate that resistance to HER2-targeted drugs variesaccording to the copy number/expression of the MEL-18 gene, and thus thedrug resistance may be inhibited or improved by amplifying or activatingMEL-18 to improve resistance of subjects with the loss or reducedexpression or activity of the MEL-18 gene, or inhibiting theamplification, expression or activity of ADAM10 and/or ADAM17.Therefore, as the present invention newly reveals that resistance toHER2-targeted drugs may be improved or inhibited by inhibition of ADAM10and/or ADAM17, and suggests a novel therapeutic approach that can beco-treated with HER2-targeted drugs, the ADAM10 and/or ADAM17inhibitor(s) or activator(s) may be widely included in the presentinvention without limitation to a specific type of agent such as acompound, an antibody or the like.

In a specific example, the ADAM10 and/or ADAM17 protein activityinhibitor(s) may be one or more selected from the group consisting ofINCB3619, INCB7839, WAY-022, TMI-2, CGS 27023, GW280264, INCB8765 andGI254023, but the present invention is not limited thereto.

INCB3619 and INCB7839 are dual inhibitors (Incyte Corporation,Wilmington, DE), which can inhibit both ADAM10 and ADAM17, and WAY-022(Wyeth-Aherst, Pearl River, NY), TMI-2 or PF-5480090 (Pfizer) is anADAM17 inhibitor. GI254023 is an ADAM10 inhibitor (Glaxo Smith Kline),and GW280264 is an ADAM10/ADAM17 inhibitor (Glaxo Smith Kline). Specificdetails of each of the above inhibitors can be found with reference tocontents known in the technical field of the present invention, forexample, those described in [Duffy et al, Clin Proteomics. 2011 Jun. 9;8(1):9].

INCB3619 is methyl(6S,7S)-7-(hydroxycarbamoyl)-6-(4-phenyl-3,6-dihydro-2H-pyridine-1-carbonyl)-5-azaspiro[2.5]octane-5-carboxylate(Incyte), which may be represented by Formula 1 below and has CAS No.791826-72-7. It is a dual inhibitor that can inhibit both ADAM10 andADAM17. According to the present invention, when subjects withoutamplification of the MEL-18 gene or subjects with low expression of theMEL-18 gene are co-treated with INCB3619, resistance to HER2-targeteddrugs may be improved, and a therapeutic effect of HER2-targeted drugsmay be enhanced.

INCB7839, also called aderbasib, is(6S,7S)-7-[(hydroxyamino)carbonyl]-6-[(4-phenyl-1-piperazinyl)carbonyl]-5-azaspiro[2.5]octane-5-carboxylicacid methyl ester (Incyte), which is represented by Formula 2 below andhas CAS No. 7918228-58-5. INCB7839 is also a dual inhibitor that caninhibit both of ADAM10 and ADAM17. Although it has been reported thatINCB7839 improves a clinical therapeutic effect of Herceptin in a groupof HER2+ metastatic breast cancer patients expressing p95-HER2, theclinical trial has been reported to have failed. However, according tothe present invention, when subjects without amplification of the MEL-18gene or subjects with low expression of the MEL-18 gene are co-treatedwith INCB7839 and HER2-targeted drugs, resistance to the HER2-targeteddrug may be improved, and the therapeutic effect of the HER2-targeteddrug may be enhanced.

WAY-022 (Wyeth-Aherst, Pearl River, NY) or TMI-2 (or PF-5480090; Pfizer)is an ADAM17-selected inhibitor, and PF-5480090 is represented byFormula 3 below. When an individual without the amplification of theMEL-18 gene or an individual with the low expression of the MEL-18 geneis treated with HER2-targeted drugs as well as WAY-022 or TMI-2(PF-5480090) according to the present invention, resistance toHER2-targeted drugs may be improved, and the therapeutic effect of theHER2-targeted drug may be enhanced.

GW280264, also known as GW280264X, has a compound name of benzylN-[(5S)-5-[[(2R,3S)-3-(Formylhydroxyamino)-2-(2-methylpropyl)-1-oxohexyl]amino]-6-oxo-6-(2-thiazolylamino)hexyl]-carbamate,which is represented by Formula 4 below and has CAS No. 866924-39-2.GW280264 may inhibit ADAM17. When an individual without amplification ofthe MEL-18 gene or an individual with low expression of the MEL-18 geneis co-treated with GW280264 as well as HER2-targeted drugs according tothe present invention, resistance to the HER2-targeted drug may beimproved, and the therapeutic effect of the HER2-targeted drug may beenhanced.

GI254023, also known as GI254023X, is(2R)—N-[(1S)-2,2-dimethyl-1-[(methylamino)carbonyl]-propyl]-2-[(1S)-1-[formyl(hydroxy)amino]ethyl]-5-phenylpentanamide, which may berepresented by Formula 5 below. GI254023 is an ADAM10-selectedinhibitor, and has about 100-fold selectivity to ADAM10 with respect toADAM17. When an individual without amplification of the MEL-18 gene oran individual with low expression of the MEL-18 gene is co-treated withGI254023 as well as HER2-targeted drugs, resistance to the HER2-targeteddrug may be improved, and a therapeutic effect of the HER2-targeted drugmay be enhanced.

INCB8765 (Incyte Corporation) is a small molecule containing ahydroxamate moiety, which has a compound name of(1R,3S,4S)-3-[(hydroxyamino)carbonyl]-4-[(4-phenylpiperidin-1-yl)carbonyl]cyclohexylpyrrolidine-1-carboxylate. INCB8765 may inhibit ADAM10 via azinc-binding mechanism. Accordingly, when an individual withoutamplification of the MEL-18 gene or an individual with low expression ofthe MEL-18 gene is co-treated with INCB8765 as well as HER2-targeteddrugs according to the present invention, resistance to theHER2-targeted drug may be improved, and the therapeutic effect of theHER2-targeted drug may be enhanced.

In one exemplary embodiment of the present invention, when GW280264inhibiting both ADAM10 and ADAM17 in a HER2+ breast cancer patient isco-administered with trastuzumab, it was confirmed that resistance totrastuzumab is improved and resistance induction is inhibited inMEL-18-depleted BT474 cells, resulting in improving an anti-tumor effectby trastuzumab. In addition, specific signaling systems for activitiesof MEL-18 and ADAM10/17 were confirmed.

In such an aspect, the composition of the present invention may beco-administered simultaneously or sequentially with HER2-targeted drugs.

The term “co-administration” or “co-treatment” used herein meansadministration of two or more types of drugs together. Theco-administration includes simultaneous or sequential administration oftwo or more drugs. When the pharmaceutical composition of the presentinvention is administered in combination with the HER2-targeted drug,resistance induced by the inhibitor may be improved, the generation ofresistance by the inhibitor may be inhibited, and the effect of aconventional therapeutic agent may also be improved.

The present invention also provides a composition for screening a drugthat improves resistance to HER2-targeted drugs or is co-administeredwith HER2-targeted drugs, which includes one or more selected from thegroup consisting of MEL-18 gene, MEL-18 protein, ADAM10 gene, ADAM10protein, ADAM17 gene or ADAM17 protein.

The present invention also provides a method of screening a drug forimproving resistance to HER2-targeted drugs or which is co-administeredwith HER2-targeted drugs, which include: contacting candidate materialswith one or more selected from the group consisting of MEL-18 gene;MEL-18 protein; ADAM10 gene; ADAM10 protein; ADAM17 gene and ADAM17protein; and selecting a candidate increasing the copy number of theMEL-18 gene; the mRNA expression level of the MEL-18 gene; or theexpression level of a protein expressed therefrom, or inhibiting themRNA expression level of ADAM10 and/or ADAM17 gene(s) or the expressionlevel of a protein expressed therefrom.

As described above, when the expression level of the MEL-18 gene orprotein is improved or the ADAM10 and/or ADAM17 protein or gene is/areinhibited, the activation of ErbB signaling inducing resistance toHER2-targeted drugs may be inhibited, and thus a material promoting theexpression level of the MEL-18 gene or protein or a material inhibitingthe ADAM10 and/or ADAM17 protein or gene may be selected as a candidatematerial of a medicine for inhibiting or improving resistance toHER2-targeted drugs.

Confirmation of the reaction between a protein or mRNA and a candidatematerial may use conventional methods used to confirm protein-protein,protein-compound, DNA-DNA, DNA-RNA, DNA-protein, DNA-compound,RNA-protein, and RNA-compound reactions. For example, a hybridizationtest for confirming in vitro binding between the MEL-18 gene and acandidate material, a method of measuring the expression rate of thegene through Northern blotting following the reaction between mammaliancells and a test material, quantitative PCR or quantitative real-timePCR, a method of introducing the gene into cells by linking a reportergene, reacting the gene with a test material, and then measuring theexpression rate of a reporter protein, a method measuring activity afterreacting the MEL-18 protein with a candidate material, yeast two-hybrid,searching for a phage-displayed peptide clone binding to the MEL-18protein, high throughput screening (HTS) using natural substance andchemical libraries, drug hit HTS, cell-based screening, or a screeningmethod using DNA array may be used.

The composition for screening may include distilled water or bufferwhich stably maintains the structure of a nucleic acid or protein, inaddition to the active ingredient. In addition, the composition forscreening may include, for an in vivo experiment, cells expressingMEL-18, or cells containing a plasmid expressing MEL-18 in the presenceof a promoter capable of regulating a transcription rate. In addition,the composition for screening may further include Dvl according to amethod of confirming the reaction between candidate materials, inaddition to the active ingredient.

In the screening method of the present invention, a test material may bean individual nucleic acid, protein, extract, natural substance orcompound which is assumed to have potential as a drug for inhibiting orimproving resistance to HER2-targeted drugs according to a conventionalselection method or randomly selected.

The present invention also provides a method of treating subjects withHER2-positive cancers, which includes administering a pharmaceuticalcomposition including one or more selected from the group consisting ofMEL-18 gene, MEL-18 gene expression activators, MEL-18 proteins, MEL-18protein activators, ADAM10 gene inhibitors, ADAM10 protein activityinhibitors, ADAM17 gene inhibitors and ADAM17 protein activityinhibitors to subjects with HER2-positive cancers, wherein subjects havethe copy number of MEL-18 gene, the mRNA expression level of the MEL-18gene, and/or the expression level of MEL-18 protein, which are/is lowerthan predetermined value(s); and/or subjects have or expected to havedrug resistance to HER2-targeted drugs.

All descriptions regarding the present invention may also be appliedmutatis mutandis to the treating method described above.

The treating method may further include co-administering HER2-targeteddrugs to subjects receiving administration of the pharmaceuticalcomposition.

In addition, the treating method may further include measuring the copynumber of the MEL-18 gene, the mRNA expression level of the MEL-18 gene,or the expression level of a protein encoded by the MEL-18 gene from asample obtained from subjects with HER2-positive cancers beforeadministration of the pharmaceutical composition. This step is forcompanion diagnostics for HER2-targeted drugs by measuring MEL-18 as acompanion diagnostic marker for the HER2-targeted drug.

The administration may include all methods for oral administration orparenteral administration, and may be performed by a methodconventionally used in the art to which the present invention belongs.In addition, in co-administration, a first drug may be administered byparenteral administration, a second drug may be administered by oraladministration, and thus, the drugs may be administered throughdifferent routes.

In the present invention, when ADAM10 and 17 inhibitors areco-administered with trastuzumab to subjects with HER2+ breast cancerand having resistance to HER2-targeted drugs, it was confirmed throughexemplary embodiments that resistance to trastuzumab is improved, andthe therapeutic effect of trastuzumab is further enhanced.

In addition, the present invention provides a use of an agent formeasuring the copy number of MEL-18 gene, the mRNA expression level ofthe MEL-18 gene, and/or the expression level of MEL-18 protein from asample obtained from subjects with HER2-positive cancers for companiondiagnostics for the HER2-targeted drug.

In addition, the present invention provides a use of an agent formeasuring the copy number of MEL-18 gene, the mRNA expression level ofthe MEL-18 gene, and/or the expression level of MEL-18 protein toprepare a composition for diagnosing subjects with HER2-positivecancers, wherein subjects have the copy number of MEL-18 gene, the mRNAexpression level of the MEL-18 gene, and/or the expression level ofMEL-18 protein, which are/is lower than predetermined value(s); and/orsubjects have or expected to have drug resistance to HER2-targeteddrugs.

In addition, the present invention provides a use of an agent formeasuring the copy number of MEL-18 gene, the mRNA expression level ofthe MEL-18 gene, and/or the expression level of MEL-18 protein toprepare a composition for companion diagnostics for HER2-targeted drugsfor subjects with HER2-positive cancers, wherein subjects have the copynumber of MEL-18 gene, the mRNA expression level of the MEL-18 gene,and/or the expression level of MEL-18 protein, which are/is lower thanpredetermined value(s); and/or subjects have or expected to have drugresistance to HER2-targeted drugs.

In addition, the present invention provides a use of an agent formeasuring the copy number of MEL-18 gene; the mRNA expression level ofthe MEL-18 gene; or the expression level of MEL-18 protein from a sampleobtained from subjects with HER2-positive cancers for preparing a kitfor companion diagnostics for HER2-targeted drugs.

In addition, the present invention provides an application of one ormore selected from the group consisting of MEL-18 gene, MEL-18 geneexpression activators, MEL-18 proteins, MEL-18 protein activators,ADAM10 gene inhibitors, ADAM10 protein activity inhibitors, ADAM17 geneinhibitors and ADAM17 protein activity inhibitors to prepare drugs fortreating subjects with HER2-positive cancers, wherein subjects have thecopy number of MEL-18 gene, the mRNA expression level of the MEL-18gene, and/or the expression level of MEL-18 protein, which are/is lowerthan predetermined value(s); and/or subjects have or expected to havedrug resistance to HER2-targeted drugs.

All descriptions regarding the present invention may also be appliedmutatis mutandis to the applications of the present invention.

Hereinafter, the present invention will be described in detail withreference to examples and experimental examples. The following examplesand experimental examples are merely provided to exemplify the presentinvention, and are not intended to limit the scope of the presentinvention.

EXAMPLES

Materials and Experimental Methods

1. Fluorescence In Situ Hybridization

Primary human breast cancer acquired from 230 breast cancer patients(HER2-positive cases, n=54; HER2− cases, n=176) was used for experimentsat Hanyang University Medical Center from January 2000 to December 2009under the approval of the Institutional Review Board of HanyangUniversity.

To treat a protein-degradable slide before hybridization, a section wasdeparaffinated, air-dried, dehydrated and denatured in a 70%formamide-2×SSC solution at 74° C. for 5 minutes. The resulting sectionwas hybridized with a selected DNA probe (PCGF2 FISH probe red 5-ROXdUTP, green 5-fluorescein dUTP chromosome 17 control probe; EmpireGenomics, Buffalo, NY, USA) in a humid chamber at 37° C. overnight,washed, and counterstained with 0.2 μmol/L DAPI in a antifade solution.Gene signals per cell were evaluated in 50 tumor nuclei for each TMAcore. The average gene copy number and the gene:CEP ratio werecalculated separately for each core, and the gene was consideredamplified when the average gene copy number was more than 6.0 (>6.0).Ambiguous cases were independently scored by two pathologists (SEL andWSK).

In addition, 213 HER2-positive invasive breast cancer tissues ofpatients receiving trastuzumab treatment and surgically resected atKonkuk University Medical Center from January 2010 to December 2016 wereenrolled under the approval of the Institutional Review Board of KonkukUniversity Medical Center (Seoul, Korea) for experiments. Consent hadbeen previously acquired from the patients. After manufacturing tissuemicroarrays, sections were deparaffinated, dehydrated and denatured.After overnight hybridization at 37° C. in a humid chamber with DNAprobes (PCGF2 FISH probe red 5-ROX dUTP, green 5-fluorescein dUTPchromosome 17 control probe; Empire Genomics, Buffalo, NY, USA), theslides were counterstained with 0.2 μmol/L DAPI. Gene signals per cellwere evaluated in 50 tumor nuclei for each case: the average gene copynumber and the gene: CEP ratio were separately calculated for eachnucleus, and the tumor was considered amplified when the average genecopy number was higher than 6.0.

2. Cell Culture and Drugs

All cells were purchased from American Type Culture Collection (ATCC,Manassas, VA, USA). Human breast cancer cells were cultured in anRPMI-1640 medium (Welgene, Daegu, Korea) containing 10% fetal bovineserum (FBS). The 293T cells were cultured in DMEM (Welgrene)supplemented with 10% FBS. All cells were incubated at 37° C. in ahumidified 5% CO₂ incubator. Trastuzumab (Herceptin) was purchased fromRoche (Basel, Switzerland), and GW280264 was obtained from Aobious(Gloucester, MA, USA).

3. Antibodies

Antibodies used in the experiments of the present invention werecommercially purchased: MEL-18(H-115, sc-10744X), phospho-EGFR (Tyr1173,sc-12351), EGFR (1005, sc-03), HER2(3B5, sc-33684)(H-224, sc-9001),HER3(C-17, sc-285), HES1(H-140, sc-25392), RING1B (N-32, sc101109),Polymerase II (H-224, sc-9001), normal mouse IgG (sc-2025), normalrabbit IgG (sc-2027), goat anti-mouse IgG-HRP (sc-2005), goatanti-rabbit IgG-HRP (sc-2030), and donkey anti-goat IgG-HRP (sc-2020)were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA);beta-actin (C4, MAB1501R), phospho-HER2(Tyr1248, 06-229), H3ac (06-599),and H3K27me3(07-499) were purchased from Merck Millipore (Billerica, MA,USA); phospho-HER3(Tyr1289, 4791S), phospho-AKT (D9E, 4060S), AKT(92725), EZH2(D2C9, 5246S), and H2AK119ub (D27C4, 8240S) were purchasedfrom Cell Signaling Technology (Beverly, MA, USA); CBX7(ab21873) andADAM10(ab1997) were purchased from Abcam (Cambridge, UK); and ADAM17(C2C3, GTX101358) was purchased from GeneTex (Irvine, CA, USA).

4. Production of Stable Cells

To establish MEL-18-overexpressing cell lines, 293T cells wereco-transfected with pLVX-MEL-18 or an empty vector construct using theLipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) as describedin [J. Y. Lee et al, J Clin Invest 125, 1801-1814(2015)] (15). After 60hours of transfection, the medium containing the lentiviruses washarvested, and transduced into target cells using 6 μm/ml polybrene(Sigma-Aldrich). To establish MEL-18-knockdown cell lines, lentivirusesencoding a GIPZ Human Lentiviral MEL-18 shRNA Clone Gene Set(RHS4531-EG7703-V3LHS334758; 5′-AGACAAUGUCUUGAAGUGU-3′; SEQ ID NO: 25)were purchased from GE Dharmacon (Lafayette, CO, USA), generated, andthen infected into cells as described above.

5. Cell Viability Test

An in vitro toxicity test analysis kit (Sulforhodamine B based, TOX6)was purchased from Sigma-Aldrich (St. Louis, MO, USA), and usedaccording to the manufacturer's protocols. Cells (3×10³ cells/well) werecultured in a RPMI 1640 medium (Welgene) supplemented with or without 10μm/ml trastuzumab, 1 μM GW280264, PBS and DMSO in a 96-well cell cultureplate, and after three days, the medium was exchanged for fresh mediumcontaining a drug. After five days, the cells were fixed by layering aTCA solution in the medium, and incubated at 4° C. for 1 hour. The fixedcells were washed with water several times, and stained with a 0.4%sulforhodamine B solution for 30 minutes. Subsequently, the stainedcells were washed with a 1% acetic acid solution until remaining saltswere no longer observed, and the incorporated dye was dissolved in a 10mM Tris solution while gently stirring. The prepared plate was detectedusing a microplate reader at 490 nm.

6. Colony Formation Assay

Cells (1×10⁴ cells/well) were treated with or without 10 μm/mltrastuzumab, 1 μM GW280264 and vehicles for 1 week in a 24-well cellculture plate, and the medium was exchanged for a fresh medium every 3days. After 2 weeks, the cells were fixed with a 4% formaldehydesolution for 5 minutes, washed with PBS, and stained with 0.02% crystalviolet (C0775, Sigma-Aldrich) for 1 to 2 hours at room temperature andthen washed again. The prepared plates were dried thoroughly, and theexperiment was carried out.

7. Real-Time Quantitative PCR

Total RNA was isolated using the TRIzol reagent (Invitrogen) and reversetranscription-PCR (RT-PCR) was performed with an Access RT-PCR system(Promega, Madison, WI, USA) according to the manufacturer'sinstructions. To measure a RNA expression level, quantitative RT-PCR(qRT-PCR) was performed using an Applied Biosystems 7300 Real-Time PCRsystem using SYBR Green dye (Applied Biosystems, Foster City, CA, USA)as described in the manufacturer's instructions. Data was normalizedwith GAPDH expression.

Primers used in this experiment are as follows:

MEL-18, (SEQ ID NO: 1) 5′-GTACTTCATCGACGCCACCACTATC-3′ and (SEQ ID NO: 2) 5′-CTCGTCCTCGTACAGAACCTCCA-3′; ADAM10, (SEQ ID NO: 3)5′-GCTACGGGCACAAGTGACTA-3′ and (SEQ ID NO: 4)5′-AGACGTAAGCAGAAACCAGACA-3′; ADAM17, (SEQ ID NO: 5)5′-AGCTCCAAAACTGGACCACC-3′ and (SEQ ID NO: 6)5′-GCTGCTATTTGGGAAGGGGT-3′; BCL-2, (SEQ ID NO: 7)5′-GAGACAGCCAGGAGAAATCA-3′ and (SEQ ID NO: 8)5′-CCTGTGGATGACTGAGTACC-3′; TGF-alpha, (SEQ ID NO: 9)5′-TCAGTCAATTTGGCCGGGAT-3′ and (SEQ ID NO: 10)5′-GGGCAGGTTGGAAGAGATCA-3′; S100A9, (SEQ ID NO: 11)5′-ACACATCATGGAGGACCTGG-3′  and (SEQ ID NO: 12)5′-TCACCCTCGTGCATCTTCTC-3′; IGFBP3, (SEQ ID NO: 13)5′-AACTGTGGCCATGACTGAGG-3′ and (SEQ ID NO: 14)5′-AGTCTCCCTGAGCCTGACTT-3′; MDK, (SEQ ID NO: 15)5′-CTCCCGGAAAGGCACTGG-3′ and (SEQ ID NO: 16) 5′-CACCTGGGGCGGTTTCC-3′;CMTM3, (SEQ ID NO: 17) 5′-GTATCTGGGCAGCAGGTGTT-3′ and (SEQ ID NO: 18)5′-ACCAAGTGCAGACAAACCCA-3′; RALGAPA2, (SEQ ID NO: 19)5′-CACTCGAATGCCGTCAGACT-3′ and (SEQ ID NO: 20)5′-TGCGGTAGTCTCTGGAGTGT-3′; USP48, (SEQ ID NO: 21)5′-GGCCGTCTGTCTCTTGGTATT-3′ and (SEQ ID NO: 22)5′-TGCACCAAAGCAAAAAGCCT-3′; and GAPDH, (SEQ ID NO: 23)5′-GAAGGTGAAGGTCGGAGTC-3′ and (SEQ ID NO: 24)5′-GAAGATGGTGATGGGATTTC-3′.

8. Immunoblotting and Immunoprecipitation

Cells were lysed in RIPA buffer, and the lysates were subjected toimmunoblotting and immunoprecipitation as described in [M. H. Cho et al,Nat Commun 6, 7821(2015)] (60). For immunoblotting, the cell lysateswere separated by 6% to 15% SDS-polyacrylamide gel electrophoresis(PAGE) and transferred to nitrocellulose membranes. After blocking themembranes with 5% skin milk, the membranes were incubated with primaryantibodies overnight at 4° C., and then reacted with horseradishperoxidase (HRP)-conjugated secondary antibodies for 1 hour at roomtemperature. Bands were visualized using an ECL detection system (GEHealthcare, Chalfont St. Giles, UK).

For immunoprecipitation, cell lysates (1 to 3 mg) were incubated withsuitable antibodies (1 μm) overnight at 4° C. The samples were incubatedagain with protein agarose A or G (20 μL) for 2 hours at 4° C. andwashed with pre-cooled PBS three times, and then 20 μL ofβ-mercaptoethanol-added 5x sample loading buffer was added toimmunoprecipitated pellets. The samples were heated at 95° C. for 5minutes, and then SDS-PAGE and immunoblotting were performed asdescribed above.

9. Gene Expression Microarray Assay

To analyze different gene expression profiles between a control andMEL-18-knockdown BT474 cells, gene expression microarrays were performedas described in [J. Y. Lee et al, J Clin Invest 125, 1801-1814(2015)]according to the manufacturer's instructions using a HumanHT-12 v4Expression BeadChip (Illumina). Total RNA was isolated from the cells,cDNA and biotinylated cRNA were generated using an Illumina Total PrepRNA amplification kit (Ambion). cRNA was hybridized on a HumanHT-12 v4Expression BeadChip (Illumina), and then the chip was washed, dried andscanned with a bead array reader. Raw data was obtained usingGenomeStudio version 2011.1 software (Illumina), and a call ofpresence/absence relied on a classification method based on detection Pvalues (gene probes with detection P values of less than 0.05, present;all other genes, absent). The selected gene probe signal values werelogarithmically transformed, and normalized using the quantile method.All gene probes displaying absolute fold changes of 1.5 or more betweencomparative controls were considered differentially expressed. Thefunctional analysis of differentially expressed genes was performedusing databases for Visualization and Integrated Discovery (DAVID) (61,62) and Gene Set Enrichment Analysis (GSEA) (63, 64).

10. Use of Public Data

Two publicly available datasets, TCGA (37) and METABRIC (38), of humanbreast cancer patients were downloaded and analyzed through cBioportal(65, 66), and re-analyzed with the Python and R programming packages.The Cancer Cell Line Encyclopedia (CCLE) dataset was analyzed throughcBioportal. A clinical dataset (GSE55348)(68, 69) for HER2+ breastcancer treated with adjuvant trastuzumab was downloaded from the GEOdatabase (www.ncbi.nlm.nih.gov/geo), and re-analyzed (Probe ID used:PCGF2, ILMN 1809859; ADAM10, ILMN 1718946; ADAM17, ILMN 1765779). Forthe analysis of the relationship between MEL-18 amplification/mRNAexpression and the OS and DFS of breast cancer patients, theKaplan-Meier estimation method was used. A public dataset (GSE67868)(67) of Mel-18 ChIP-seq in mouse embryonic stem cells was obtained fromGEO for re-analysis of MEL-18 protein-binding genes.

11. Chromatin Immunoprecipitation (ChIP) Assay

A ChIP assay was performed as described in [M. H. Cho et al, Nat Commun6, 7821(2015)] (60). Cells were crosslinked with 1% formaldehyde at roomtemperature for 10 minutes, lysed in 200 μL of SDS lysis buffer, andsonicated using a Bioruptor (Cosmo Bio Co. Ltd, Tokyo, Japan). The celllysate was incubated with specific antibodies overnight at 4° C., andthen incubated with protein A- or G-agarose coupled to salmon sperm DNA(Millipore) for additional 2 hours. The immunoprecipitates were washed,eluted and reverse-crosslinked with 20 μL of 5M NaCl at 65° C.overnight. DNA fragments precipitated from the eluate were used forreal-time quantitative PCR (qPCR) to analyze the intensity of a ChIPsignal.

ChIP-qPCR was performed using the following specific primers: ADAM10primer, 5′-GCTCGGAAAATTACATCTCGGAC-3′ (SEQ ID NO: 26) and5′-GCCCCAGTGATGCGAACATA-3′ (SEQ ID NO: 27); and ADAM17 primer,5′-GCCGCTTTCTACAGCTCCTTT-3′ (SEQ ID NO: 28) and5′-GCCTCCTTTGTCTTGATGCC-3′ (SEQ ID NO: 29).

12. Phospho-Receptor Tyrosine Kinase (RTK) Assay

Kinase activity was detected in an RTK panel using a human phospho-RTKarray kit (R&D Systems). Cells were harvested according to themanufacturer's protocol, washed with cold PBS and lysed with lysisbuffer, and then a 100 to 200 μg lysate was incubated with a blockedmembrane overnight at 4° C. The membrane was exposed to achemiluminescent reagent, an image was captured, and quantification wasperformed using AlphaEaseFC software (Alpha Innotech, Inc., San Leandro,CA, USA).

13. Enzyme-Linked Immunospecific Assay (ELISA)

The levels of the HER ligands HB-EGF and heregulin1 (heregulin1,NRG1-beta1, NRG1) were measured in cell culture supernatants using ELISA(BosterBio and R&D Systems) according to the manufacturer's protocols.

In the case of the R&D Systems kit, an ELISA plate was coated overnightwith captured antibodies and blocked with 3% BSA for 1 hour.Subsequently, 100 μL of samples and standards were loaded, and the platewas maintained at room temperature for 2 hours. The plate wells werewashed and incubated with detection antibodies for 2 hours, and thenincubated with streptavidin labeled with horseradish peroxidase for 20minutes.

In the case of the BosterBio ELISA kit, the antibody-coated plate wasdirectly filled with samples and standards, and incubated for 90minutes. Subsequently, the samples were removed, and the plate wasincubated with biotinylated anti-human HB-EGF antibodies for 60 minutesand washed. The plate was incubated with an ABC solution for 30 minutesand washed. After this step, the plates for both kits were filled with asubstrate solution for 20 minutes, the reaction was stopped using a stopsolution for 20 minutes, and the absorbance was measured using amicroplate reader at a wavelength of 450 nm. Each incubation for theBosterBio ELISA kit was performed at 37° C.

14. Mouse Xenograft Model

All animal experiments were approved by the Hanyang University AnimalCare and Use Committee (Seoul, Korea). Four-week-old female NOD/SCIDmice were purchased from Koatech (Pyeongtaek, Gyeonggi). For orthotopicxenografts, control and MEL-18-knockdown BT474 cells (2×106) wereresuspended in 150 uL of a 1:1 mixture of PBS/Matrigel, and directlyinjected into breast fat pads of the mice (left, control; right, MEL-18shRNA). One day before the cell injection into the mice, estradiolpellets (0.72 mg, 60-day release; Innovative Research of America,Sarasota, FL, USA) were provided. After a tumor size reached 100 mm³,the mice were treated twice weekly with either a vehicle (PB S) ortrastuzumab (20 mg/kg) by intraperitoneal injection. The tumor size wasmeasured twice weekly, and the mice were monitored daily for clinicalsigns of toxicity during a treatment period.

The tumor size was calculated as follows: Volume (mm³)=(a×b²)/2, whereina is the largest diameter, and b is a vertical diameter.

15. Immunohistochemistry

Formalin-fixed and paraffin-embedded tumor sections from xenograftedmice were deparaffinized, rehydrated, and immunostained with suitableprimary antibodies against MEL-18 (1:25), Ki-67 (1:150), ADAM10 (1:100),and ADAM17 (1:100) using an automated system, Leica Bond III (LeicaBiosystems, Nusslock, Germany) with a Bond Polymer Refine Detection Kit(Leica Biosystems). Expression was graded according to the intensity andpercentage of positive staining of tumor cells as follows: Intensity, 0(no staining), 1 (weak), 2 (moderate) and 3 (strong); 0 (0%-5%), 1(6%-25%), 2 (26%-50%), 3 (51%-75%) and 4 (>75%); the extent of staining,0 (0%-5%), 1 (6%-25%), 2 (26%-50%), 3 (51%-75%), and 4 (>75%). To obtaina combined immunoreactive score (IRS), the score for intensity wasmultiplied by the score for extent. For survival analysis of breastcancer patients treated with trastuzumab (n=213) according to ADAM10 orADAM17 expression, immunohistochemical analysis was performed asdescribed above. When the intensity score was 2 or more, the expressionstatus was considered positive.

16. Statistical Analysis

The statistical significance of the difference between a control and anexperimental group was determined using the two-tailed Student's t-test.For multiple group comparison, following Welch ANOVA, a post-hoc LSDtest for equal variances following non-equal distribution or one-wayANOVA was performed with the Dunnett's T3 test. Pearson's correlationwas used for analysis of the correlation between MEL-18 expression andits amplification or HER2 expression. The Kaplan-Meier curves for OS andDFS analyses were evaluated using a long-rank test. These analyses wereperformed using SPSS software (ver. 12.0), the Python statisticalpackage (www.python.org/) and the R statistical package(www.r-project.org/) or the Excel (Microsoft, Redmond, WA, USA) softwarepackage. In all cases, P values of less than 0.05 (P value <0.05) wereconsidered statistically significant.

EXPERIMENTAL EXAMPLES: RESULTS

1. Clinical Meaning of MEL-18 Amplification in HER2-Positive BreastCancer

Previous studies of the inventors have supported various tumorsuppressive functions of MEL-18 in human breast cancer (4, 11-15), andindicate that MEL-18 loss is indicative of aggressive phenotypeacquisition in breast cancer. To further define the significance ofMEL-18 expression in human cancer, the genetic abnormality of PCGF2,which is a gene encoding MEL-18 located at 17q21 in breast cancer, andthe genetic aberration of the MEL-18 gene were investigated in multiplebreast cancer cohorts. In the Cancer Genome Atlas (TCGA; 37) andMETABRIC datasets (38), there were low rates of deletion and no somaticmutations found in the MEL-18 gene, and in basal-like breast cancer,downregulation of a MEL-18 mRNA level was consistent with the previousstudies (15) (FIG. 1A). Notably, the amplification of the MEL-18 geneoccurred exclusively in HER2-amplified breast cancer, accounting forapproximately half (FIG. 1A and Table 1).

TABLE 1 MEL-18 expression Cohorts HER2 expression Positive Negative HYUPositive 23 cases 31 cases (n = 230) (n = 54) (42.6%) (57.4%) Negative 0case 176 cases (n = 176) (0%) (90%) TCGA Positive 47 cases 58 cases (n =817) (n = 105) (44.8%) (55.2%) Negative 2 cases 710 cases (n = 712) (6%)(94%) METABRIC Positive 146 cases 152 cases (n = 1980) (n = 298) (49.0%)(51.0%) Negative 4 cases 1678 cases (n = 1682) (0.2%) (99.8%)

In FISH analysis of the cohorts, almost a half of the HER2-positivebreast cancer patients exhibited MEL-18 amplification (FIG. 1A). Otherthan breast cancer, according to HER2-positive status, similar rates ofMEL-18 amplification occurred in other types of cancer (FIG. 7A). Thefrequency of the MEL-18 gene amplification in the 17q12-21 amplicon wasdependent on the distance from the HER2 gene (FIG. 7B). From TCGA andMETABRIC datasets, it was confirmed that MEL-18 gene amplification wasclosely associated with the increased MEL-18 mRNA expression level inbreast cancer patients (FIG. 1B). In addition, the positive correlationbetween MEL-18 and HER2 expression was confirmed from these cohorts(FIG. 1C), and MEL-18 expression was higher in the HER2 subtype,compared to other subtypes (FIG. 1C). In the survival analysis ofHER2-positive breast cancer patients using METABRIC datasets,MEL-18-amplified patients had much higher overall survival (OS) anddisease-free survival (DFS) rates (P=0.013 and P=0.023, respectively;FIG. 1D). In addition, MEL-18 amplification or high MEL-18 expressionwas associated with favorable DFS in patients receiving trastuzumabtreatment. Collectively, such result supported the clinical significanceof MEL-18 in HER2-positive breast cancer as an indicator of a favorablesurvival outcome associated with a response to anti-HER2 therapy.

2. MEL-18 Amplification-Induced Trastuzumab Sensitivity in HER2-PositiveBreast Cancer

Based on previous studies supporting the role of MEL-18 knockdown in theregulation of CSC and EMT associated with resistance to an anticanceragent (13, 14), MEL-18 is likely to be involved in a response totrastuzumab, and is estimated to affect the prognosis of HER2-positivebreast cancer. To confirm this, an experiment was performed.

To confirm the functional relevance of MEL-18 amplification inHER2-positive breast cancer, genetic variation and mRNA levels of MEL-18were examined in various types of HER2-positive breast cancer celllines. Half of HER2-positive cell lines in the Cancer Cell LineEncyclopedia (CCLE) dataset, consistent with clinical data, exhibitedMEL-18 amplification and mRNA expression correlated therewith, which isthe same as confirmed by FISH and qRT-PCR analysis (FIG. 2A and FIG.2B).

Afterward, in respective various MEL-18-amplified andMEL-18-nonamplified, HER2-positive breast cancer cell lines (BT474,ZR-75-30, SKBR2 and HCC-1419), lentiviral MEL-18 knockdown andoverexpression systems (FIG. 2C), and responses of these cells totrastuzumab treatment were compared.

As shown in FIGS. 2C, 2D and 2E, similar to the results obtained by invitro cell viability analysis, the shRNA-mediated knockdown of MEL-18induced trastuzumab resistance, whereas MEL-18 overexpression increasedtrastuzumab sensitivity in these cells (FIG. 2C). Specifically,shRNA-mediated inhibition of MEL-18 in BT474 cells induced trastuzumabresistance, whereas MEL-18-overexpressing SKBR3 cells exhibited a highergrowth inhibitory effect than trastuzumab-treated control cells. InHCC-1954 cells having trastuzumab resistance due to I3KH1047R mutation,MEL-18 overexpression did not make the cells sensitive to trastuzumab,but brought a considerable decrease in cell growth (FIG. 2D). Like thein vitro results, although orthotopic mouse xenograft models ofMEL-18-knockdown BT474 cells exhibited increased promoted tumor growthregardless of trastuzumab treatment, control mice responded totrastuzumab (FIGS. 2E and 2F). Such a result shows that MEL-18 lossmediates trastuzumab resistance, and indicates that MEL-18 amplificationcan be a predictor for trastuzumab sensitivity in HER2-positive breastcancer.

3. Confirmation of MEL-18 Effect on Activation of ErbB Family Receptor

Next, the role of MEL-18 in regulation of trastuzumab responses inHER2-positive breast cancer was examined, and the result is shown inFIGS. 3A, 3B, 3C, 8A and 8B.

Gene expression microarray analysis showed different gene expressionprofiles between a control and MEL-18-knockdown HER2-positive breastcancer cells (FIG. 3A), and here, gene sets associated with EGFRsignaling and growth factor activity, and different oncogenesis pathwayswere significantly increased (FIG. 3B, and FIGS. 8A and 8B). Changes inMEL-18 targeted gene expression involved in ADAM10, ADAM17, S100A9, TGF-and BCL-2 pathways were proved by qRT-PCR (FIG. 3C).

To further confirm whether microarray MEL-18 changes the activity of anEGFR-related signaling pathway as suggested in the microarray analysis,under MEL-18 expression, phosphorylation profiles of several RTKs weremeasured using phospho-RTK array and immunoblotting.

As shown in FIGS. 3D and 3E, in MEL-18-overexpressing SKBR3 cells,compared with control cells, the phosphorylation levels of EGFR, HER2and HER3, constituting the ErbB family, were significantly reduced, butexpression levels were not reduced (FIGS. 3D and 3E). Similar effectswere also observed in MEL-18-knockdown BT474 cells (FIG. 3E).Consequently, AKT phosphorylation was negatively regulated by MEL-18 inthese cells, indicating that the ErbB-PI3K-AKT pathway is inhibited byMEL-18 (FIG. 3E). Therefore, it was concluded that MEL-18 inhibits theregulation of an ErbB signaling pathway by changing the kinase activityof the ErbB family.

4. Confirmation of Epigenetic Suppression of MEL-18-Mediated ErbB LigandSheddase ADAM10/17

To identify the molecular mechanism by which MEL-18 affectsphosphorylation levels of ErbB family receptors, the expression of ADAMfamily sheddases for an ErbB ligand involved in ligand-dependent ErbBsignaling activity and anti-HER2 therapy resistance was furtherconfirmed.

As shown in FIGS. 4A, 4B, 4C and 9 , MEL-18 knockdown increased proteinand mRNA levels of ADAM10 and ADAM17 in BT474 cells, whereas MEL-18overexpression decreased their expression in SKBR3 cells (FIGS. 4A and4B). This means that MEL-18 plays a role in regulation of genetranscription. Chromatin immunoprecipitation (ChIP) analysis confirmedan increase in MEL-18 binding to ADAM10 and ADAM17 promoter regions inHER2-positive human breast cancer cell lines (FIG. 4C). After MEL-18binds to the promoters, transcriptional active marker RNA polymerase II(Pol II) was dissociated from regions thereof, and histone H3acetylation (H3ac) was also decreased in MEL-18-overexpressing SKBR3cells (FIG. 4C). In addition, MEL-18 changed the modification of PRC1-and PRC2-dependent histones at these regions. In PRC1 members, CBX7 andRING1B well known as MEL-18 binding partners (42, 43) were co-bonded inADAM10 and ADAM17 promoter regions, and H2AK119ub levels increaseddependently with MEL-18 expression levels. In addition, MEL-18 promotesthe binding and enzymatic activity of EZH2, which is a catalyst subunitof PRC2 with respect to H3K27 in these regions. Similarly, PRC2-inducedH3K27me3 and PRC1-mediated H2AK119ub were reduced in ADAM10 and ADAM17promoters in MEL-18-knockdown BT474 cells (FIG. 4C).

In addition, as shown in FIGS. 4D and 4E, co-immunoprecipitationanalysis clearly confirmed that MEL-18-dependent EZH2 recruitment totarget chromatin is caused by the interaction between MEL-18 and EZH2proteins (FIG. 4D). This result shows that MEL-18 epigeneticallysuppresses the expression of ErbB ligand sheddases ADAM10 and ADAM17 byforming a transcription inhibitory complex in cooperation with PRC2 andPRC1 (FIG. 4E).

5. Role of ADAM10/17 in Mediation of MEL-18 Loss-Induced TrastuzumabResistance

To verify whether changes in the expression of ADAM10 and ADAM17 byMEL-18 play a role in ligand-dependent ErbB signaling associated withtrastuzumab resistance, the effect of MEL-18 on the production of ErbBligands was investigated.

As shown in FIGS. 5A and 5B, in MEL-18-knockdown BT474 cells, secretedHB-EGF and heregulin-1 (NRG1) levels and the amounts of ligands (17)specific for EGFR and HER3 increased (FIG. 5A). In contrast, theproduction of these ligands was reduced in response to MEL-18overexpression in SKBR3 cells (FIG. 5A). In addition, the effect ofMEL-18 on production of ErbB ligands led to a change inheterodimerization between ErbB family receptors. Co-immunoprecipitationanalysis shows that MEL-18 knockdown in BT474 cells enhanced the bindingof HER2 with EGFR and HER3, and MEL-18 overexpression in SKBR3 cellsinhibits their dimerization (FIG. 5B).

In addition, as shown in FIGS. 5C and 5D, as a result of treatment withthe ADAM10/17 dual inhibitor GW280264, such effects were mediated by theMEL-18 knockdown-mediated secretion of ErbB ligands and the sheddaseactivity of ADAM10 and ADAM17 reversing the activation of ErbB-PI3K-AKTsignaling in BT474 cells (FIGS. 5C and 5D).

In addition, as shown in FIG. 5E, as a result of co-treatment ofGW280264 with trastuzumab, it was confirmed that the anti-proliferativeeffect of trastuzumab in MEL-18-depleted BT474 cells was improved (FIG.5E).

Moreover, consistent with the in vitro results, MEL-18 loss wasassociated with an increase in protein levels of ADAM10 and ADAM17 inbreast tumors from xenograft mice (FIG. 5F). Particularly, in breastcancer patients (n=213) receiving trastuzumab treatment, a subgrouphaving MEL-18 amplification and a low expression level of ADAM17 hadremarkably favorable disease-free survival (DFS), whereas a group havinghigh ADAM17 expression and non-amplification of MEL-18 was associatedwith lower DFS (left, FIG. 5G). Patients showing low ADAM10 expressionand MEL-18 amplification had a tendency toward a more favorable survivalresult. The analysis of a gene expression array from a public databasealso showed that trastuzumab-treated patients having overexpression ofMEL-18 mRNA and underexpression of ADAM10 or ADAM17 mRNA showedprolonged relapse-free survival (right, FIG. 5G).

Therefore, this data indicated that ADAM10/17 sheddases triggerMEL-18loss-induced trastuzumab resistance by mediating ErbB ligand productionand receptor activation.

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1. A method of screening drugs for improving resistance to HER2-targeteddrugs or being co-administrated with HER2-targeted drugs, the methodcomprising: contacting candidate materials with one or more selectedfrom the group consisting of MEL-18 genes, MEL-18 proteins, ADAM10genes, ADAM10 proteins, ADAM17 genes and ADAM17 proteins, and selectingthe candidate material that increases the copy number of the MEL-18gene, the mRNA expression level of the MEL-18 gene, or the expressionlevel of MEL-18 protein expressed from the MEL-18 gene; or inhibits theexpression level(s) of the ADAM10 and/or ADAM17 gene(s) or theexpression level of a protein expressed from the gene(s).
 2. The methodof claim 1, wherein the gene copy number; the mRNA expression level ofthe MEL-18 gene; or the expression level of the MEL-18 protein ismeasured by a method selected from the group consisting of fluorescentin situ hybridization (FISH), comparative genomic hybridization(CGH-based array), a single nucleotide polymorphism (SNP) array,sequence assembly comparison, paired-end sequencing, multiplex ligationdependent probe amplification (MLPA), multiplex amplifiable probehybridization (MAPH), quantitative multiplex PCR of short fluorescentfragments (QMPSF), microsatellite genotyping, Southern blotting,immunohistochemistry polymerase chain reaction (PCR), quantitative PCR(qPCR), quantitative real-time PCR (qRT-PCR), real-time PCR,microarray-based comparative genomic hybridization and ligase chainreaction (LCR).
 3. The method of claim 1, wherein the HER2-targeteddrugs are selected from the group consisting of trastuzumab, pertuzumaband trastuzumab emtansine (T-DMI).